Báo cáo khoa học: Gonadotropin-releasing hormone and ovarian cancer: a functional and mechanistic overview

M I N I R E V I E W

Gonadotropin-releasing hormone and ovarian cancer: a functional and mechanistic overview Wai-Kin So, Jung-Chien Cheng, Song-Ling Poon and Peter C. K. Leung

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada

Keywords apoptosis; G protein; GnRH; gonadotropin- releasing hormone; growth factor; invasion; MAPK; migration; ovarian cancer; proliferation

Correspondence P. C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, 2H30, 4490 Oak Street, Vancouver, BC, Canada V6H 3V5 Fax: +1 604 875 2717 Tel: +1 604 875 2718 E-mail: peleung@interchange.ubc.ca

(Received 7 May 2008, revised 5 August 2008, accepted 15 August 2008)

doi:10.1111/j.1742-4658.2008.06679.x

The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) is well known for its role in the control of pituitary gonadotropin secretion, but the hormone and receptor are also expressed in extrapituitary tissues and tumor cells, including epithelial ovarian cancers. It is hypothesized that they may function as a local autocrine regulatory system in nonpituitary contexts. Numerous studies have demonstrated a direct antiproliferative effect on ovarian cancer cell lines of GnRH and its synthetic analogs. This effect appears to be attributable to multiple steps in the GnRH signaling cascade, such as cell cycle arrest at G0 ⁄ G1. In contrast to GnRH signaling in pituitary gonadotropes, the involvement of Gaq, protein kinase C and mitogen-activated protein kinases is less apparent in neoplastic cells. Instead, in ovarian cancer cells, GnRH receptors appear to couple to the pertussis toxin-sensitive protein Gai, leading to the activation of protein phosphatase, which in turn interferes with growth factor-induced mitogenic signals. Apoptotic involvement is still controversial, although GnRH ana- logs have been shown to protect cancer cells from doxorubicin-induced apoptosis. Recently, data supporting a regulatory role of GnRH analogs in ovarian cancer cell migration ⁄ invasion have started to emerge. In this mini- review, we summarize the current understanding of the antiproliferative actions of GnRH analogs, as well as the recent observations of GnRH effects on ovarian cancer cell apoptosis and motogenesis. The molecular mechanisms that mediate GnRH actions and the clinical applications of GnRH analogs in ovarian cancer patients are also discussed.

rounds of

Gonadotropin theory of ovarian cancer

successive rupture and repair, surface increases the chance of accumulating genetic aberra- tions and therefore malignant transformation [1]. The hypothesis is supported by substantive epidemiological data. For example, one case–control study of 150 ovar- ian cancer patients under the age of 50 years demon- strated that the risk of ovarian cancer decreased with increasing numbers of live births, increasing numbers

Ovarian cancer is the most lethal gynecological malig- nancy. Although epithelial ovarian carcinomas account for approximately 90% of all human ovarian cancers, the etiology of this disease is poorly understood. Fat- halla proposed the ‘incessant ovulation theory’ in 1971, suggesting that continuous ovulation, associated with

Abbreviations AP-1, activator protein-1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; GnRH, gonadotropin-releasing hormone; GPCR, G-protein-coupled receptor; IGF-I, insulin-like growth factor-I; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; MMP, matrix metalloproteinase; NF-jB, nuclear factor kappa B; OSE, ovarian surface epithelium; PKC, protein kinase C; PLC, phospholipase C; PP2A, protein phosphatase 2A; PTP, phosphotyrosine phosphatase; PTX, pertussis toxin; TIMP, tissue inhibitor of metalloproteinases.

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extracellular

of incomplete pregnancies, and the use of oral contra- ceptives [2]. Another prevailing hypothesis addressing the development of ovarian cancer was proposed by Cramer and Welch in 1983. Their ‘gonadotropin the- ory’ proposed that excessive gonadotropin stimulation contributes to ovarian carcinogenesis [3]. The risk of ovarian cancer increases during the perimenopausal period, when serum gonadotropin levels peak and thereafter remain elevated [4,5]. Moreover, only 10– 15% of tumors appear in premenopausal women [6]. Likewise, polycystic ovary syndrome patients (with high luteinizing hormone levels) are more prone to ovarian cancer [7]. Epidemiologic evidence supports the idea that pregnancies, breast feeding, and oral contra- ceptive use, which suppress pituitary gonadotropin secretion, reduce the risk of ovarian cancer [8–11]. Experimentally, expression of gonadotropin receptors has been detected in ovarian cancer tissue and in the precursor ovarian surface epithelium (OSE) cells [12– activate mitogenic pathways, 14]. Gonadotropins including the signal-regulated kinase (ERK) pathway [14–16], and promote ovarian cancer cell proliferation [12–14] and invasion [17].

The extremely short half-life of hypothalamic GnRH makes it an unlikely candidate to act on the ovary via the systemic circulation and suggests the existence of a local source of GnRH in ovarian cancer cells. Indeed, our group and others have detected GnRH-I mRNA in normal OSE and immortalized OSE cells, as well as in primary cultures of ovarian tumors and ovarian car- cinoma cell lines such as EFO-21, EFO-27, CaOV-3, OVACR-3 and SKOV-3 [32,33]. Similarly, GnRH-II mRNA has been detected in normal and neoplastic OSE cell lines and primary cultures of ovarian carcino- mas [28]. GnRH-like immunoreactivity was detected in conditioned media [34] and cell lysates [21] from ovar- ian cancer cell lines. The latter possessed bioactivity comparable to that of authentic GnRH, as it stimu- lated luteinizing hormone release from rat pituitary [21]. Incubation of ES-2 ovarian cancer cells in vitro with a GnRH-I antibody inhibited cell proliferation in a time- and dose-dependent manner [34], whereas Emons reported a signiﬁcant increase in EFO-21 and EFO-27 ovarian cancer cell proliferation after GnRH-I [35]. Despite this discrepancy, antiserum treatment these studies provide direct evidence for the endo- genous secretion of bioactive GnRH as an autocrine growth-regulatory loop in ovarian cancer cells.

Desensitization or downregulation of the gonadotro- pin-releasing hormone (GnRH) receptors on pituitary gonadotropes by chronic administration of GnRH agonists or competitive binding of the GnRH receptors by GnRH antagonists can block gonadotropin secre- tion and subsequently suppress gonadotropin-depen- dent functions in the ovary [18]. GnRH agonists have also been shown to inhibit the growth of heterotrans- planted ovarian cancer in nude mice, presumably via altering circulating gonadotropin or steroid levels [19], but a direct effect of GnRH on the cancer cells cannot be excluded.

GnRH ⁄ GnRH receptor autocrine system in ovarian cancer cells

Our laboratory demonstrated the existence of an autocrine loop involving GnRH and the GnRH recep- tor in primary cultures of human OSE cells (scraped from the ovarian surface during laparoscopies for [32]. The GnRH agonist nonmalignant disorders) [D-Trp6]GnRH had a direct inhibitory effect on growth of OSE cells in a time- and dose-dependent manner. This inhibitory effect was reversed by cotreat- ment with the GnRH receptor antagonist antide [32]. Moreover, the GnRH agonist has a homologous regulatory effect on the expression of GnRH and the GnRH receptor in OSE cells, which further supports the presence of an autocrine regulatory GnRH system that is operational in the ovary.

Antiproliferative effect of GnRH analogs on ovarian cancer

Numerous in vitro studies have reported a growth- modulating effect of GnRH-I ⁄ GnRH-II and their synthetic analogs in various GnRH receptor-bearing lines (Table 1). In most cases, ovarian cancer cell GnRH-I and its agonists were reported to inhibit ovar- ian cancer cell proliferation, as judged by decreased cell number or DNA synthesis. For example, our labo- ratory has reported that treatment with the agonist [D-Ala6]GnRH caused a time- and dose-dependent inhibition of cell proliferation in the ovarian cancer

Expression of GnRH receptors and speciﬁc GnRH- binding sites have been detected in primary cultures of ovarian carcinomas [20] and ovarian carcinoma biopsy specimens [21,22], including mucinous and serous sub- types [23]. The widespread presence (> 80%) of GnRH-binding sites in biopsy samples [24,25] supports the involvement of a GnRH regulatory system in ovar- ian cancers. We and others have also demonstrated the presence of GnRH receptors in various established ovarian cancer cell lines, including BG-1, OVCAR-3, SKOV-3, EFO-21 and EFO-27 (Table 1) [20,26–31]. The level of GnRH receptor expression in the ovarian cancer cell lines was about 10-fold lower than that in pituitary aT3 cells [20].

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GnRH and ovarian cancer

Table 2. Overview of trials using GnRH agonists in ovarian cancer. CR, complete response; PR, partial response; SD, stable disease.

Reference

Drug

Patients

[107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [106] [119] [120]

Leuprolide acetate Leuprolide acetate Leuprolide acetate Leuprolide acetate Leuprolide acetate Leuprolide acetate Leuprolide acetate Triptorelin Triptorelin Triptorelin Triptorelin Triptorelin Triptorelin Goserelin Goserelin

18 5 25 32 32 37 12 41 19 20 40 14 68 23 30

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4 3 1 4 2 0 1 6 11 0 0 0 0 4 2

2 1 15 5 4 4 3 5 0 14 1 8 11 7 5

cell line OVCAR-3. Signiﬁcant inhibition was detected as early as 2 days after treatment [36]. Interestingly, GnRH-I antagonists consistently act like agonists and inhibit cell proliferation in various cell lines (Table 2). GnRH-I antagonists were reported to be more potent than equimolar concentrations of agonists in inhibiting ovarian cancer cell growth [29,37]. This phenomenon was also observed in endometrial [38], prostate [39] and breast cancers [40], suggesting that the dichotomy between GnRH-I agonists and antagonists in the pituitary might not be applicable to cancer cells. The exact mechanism underlying the difference between the pituitary and extrapituitary tissues remains to be elucidated.

In contrast to the situation with GnRH-I, functional studies on GnRH-II are rather limited. We and others have reported that, like GnRH-I agonists, GnRH-II and its agonists, such as d-Arg(6)-Azagly(10)-NH2, exert an antiproliferative effect on ovarian cancer cell lines [41,42]. The antiproliferative effect of GnRH-II is more potent than that of GnRH-I [37]. Interestingly, it has been reported that a GnRH-II agonist inhibits the growth of SKOV-3 cells, which are GnRH-I recep- tor-negative and unresponsive to GnRH-I [27]. The antiproliferative effect of GnRH-I is associated with an induction of cell cycle arrest at G0 ⁄ G1 [43–46], cou- pled with a downregulation of Cdk expression [44] and cyclin A–Cdk2 complex levels [46], or inhibition of telomerase activity without alteration of RNA expression [47].

In addition to cell cycle arrest, apoptosis may also be involved in the antiproliferative action of GnRH. GnRH-I agonists have been reported to induce pros- tate cancer apoptosis [48]. In ovarian cancer cells, a

high concentration (10)5 m) of GnRH agonist has been reported to induce tumor necrosis factor-a secre- tion, interchromosomal DNA fragmentation, and a marginal apoptotic effect [49]. An equimolar concen- tration of the GnRH-I antagonist cetrorelix induced apoptosis by upregulating p53 and p21 protein levels, whereas concentrations as low as 10)9 m resulted in antiproliferative effects [46]. Recently, apoptosis was shown to be induced by a low concentration of cetror- elix in ovarian cancers [50]. We also observed DNA fragmentation after prolonged (6 days) low-dose GnRH-I agonist treatment [36]. In most studies, how- ever, apoptosis was induced only when ovarian cancer cells were treated with GnRH-I analogs at relatively high concentrations or for a prolonged time. Although Fas and FasL were detected in the majority of ovarian carcinomas and ovarian cancer cell lines [51,52], and GnRH agonists such as buserelin dose-dependently induced FasL expression in ovarian cancer cells [52], a causative linkage between Fas ⁄ FasL and the antipro- liferative action of GnRH has not been established. Indeed, there is no consensus about the proapoptotic role of GnRH. The antiproliferative effect of GnRH has been mainly attributed to the cytostatic action of GnRH rather than induction of apoptosis. GnRH-I including triptorelin [44,53] and leuprolide agonists, [54], were marginally effective or ineffective in induc- ing ovarian cancer cell apoptosis. In contrast, these agonists exerted a protective effect against the cyto- toxic action of the chemotherapy drug doxorubicin [53,54]. Abolition of GnRH action by GnRH receptor knock-down increased doxorubicin-induced apoptosis [55]. A GnRH-generated protective effect against doxorubicin-induced apoptosis was also observed in human granulosa, breast cancer and endometrial cancer cells [55,56]. The underlying mechanism of this protective effect is unknown, although activation of nuclear factor kappa B (NF-jB) may be involved. Triptorelin was shown to activate NF-jB in ovarian cancer cells, and blockage of NF-jB translocation into the nucleus reversed GnRH-induced protection against doxorubicin [53] (Fig. 1F). In this case, the antitumor (antiproliferative) and antiapoptotic effects of GnRH would appear to be paradoxical, but in fact doxorubi- cin and most chemotherapy drugs are more efﬁcacious towards rapidly dividing cells, and thus the cell cycle arrest induced by GnRH can protect the tumor cell from doxorubicin. Regarding GnRH-II, we and others showed that GnRH-II and its antagonist induced ovarian cancer cell apoptosis [57,58], which was medi- ated by p38 mitogen-activated protein kinase (MAPK) and caspase-3 activation [57,58]. Furthermore, an antagonists was of GnRH-II antitumor

effect

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EGF

GnRH-I agonist/ antagonist

GnRH-I antagonist

EGFR

GnRH-I receptor

GnRH-II receptor ?

A

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T PP2A r a n s l o c a t i o n

JunD

ERK1/2

EGFR Expression

NF κ κ B

AP-1

c-fos expression

Apoptosis

Cell cycle

Proliferation

Fig. 1. GnRH-I signaling in ovarian cancer cells. (A) Through Gai, GnRH-I analogs acti- vate PTP to dephosphorylate EGFR and abolish EGF-induced ERK activation, c-fos expression and proliferation. (B) Gbc subunit activates ERK and mediates GnRH-I-induced growth inhibition. (C) GnRH-I activates ERK through a PKC-dependent or PKC-indepen- dent pathway to inhibit proliferation. (D) GnRH-I activates JNK, which increases AP-1 activity and JunD–DNA binding to extend the cell cycle. (E) GnRH-I suppresses apop- tosis through activation of PP2A. (F) GnRH-I stimulates NF-jB activity and nuclear trans- location to protect ovarian cancer cells from apoptosis. (G) GnRH-I acts through Gai to counteract forskolin (FK)-induced cAMP. The presence of a functional GnRH-II receptor has yet to be evaluated. Dashed arrows rep- resent the EGF-stimulated mitogenic signal- ing pathway; FS, forskolin; AC; adenylate cyclase.

demonstrated in nude mice bearing ovarian cancer cell xenografts [58].

further

(Fig. 2C). We

plasminogen

activator ⁄ plasminogen

stimulated the migration and invasion potential of CaOV-3 and OVCAR-3 ovarian cancer cells. The GnRH-induced increase in invasiveness and migratory activity was blocked by neutralizing antibodies against MMP-2 and MMP-9 [63]. This motogenic action of GnRH was mediated by GnRH receptor and c-Jun N-terminal kinase (JNK), but not by ERK or p38 investigated MAPK [63] the effect of GnRH-II in ovarian cancer cells. The GnRH-II agonist d-Arg(6)-Azagly(10)-GnRH-II, like GnRH-I agonists, stimulated OVCAR-3 cell invasion. Interestingly, high doses of GnRH-I and GnRH-II agonists were observed to reduce the invasive potential of SKOV-3 cells by altering the balance between MMP and TIMP [64]. In this regard, it is noteworthy that GnRH-I agonists and antagonists have been reported the migration and invasion of prostate to inhibit cancer, breast cancer and epidermoid carcinoma cells [65–67]. Also, breast cancer cell invasiveness was sup- pressed in vitro by both GnRH-I and GnRH-II [67].

Signaling and mechanism of GnRH action in ovarian cancer cells

As a member of the serpentine receptor family, the GnRH receptor transmits its signals mainly through (GTP-binding proteins). heterotrimeric G-proteins

In contrast to the relatively large number of studies on GnRH actions such as antiproliferative and apop- totic ⁄ antiapoptotic effects, reports of GnRH inﬂuences on other parameters of ovarian cancer progression, such as tumorigenic or metastatic processes, are lim- ited. Spread of ovarian cancer beyond the ovaries to the peritoneal cavity leads to later staging of the dis- ease and poor prognosis. The fact that a high propor- tion of advanced-stage (stages III and IV) ovarian express GnRH receptor mRNA and carcinomas protein, as compared to early-stage carcinomas [59], has prompted us to investigate the participation of GnRH in regulating migration and invasion in ovarian cancer. Previously, we have demonstrated the potency of GnRH-I and GnRH-II regulation of the urokinase- type inhibitor system and matrix metalloproteinase (MMP)-2, MMP- 9, and tissue inhibitor of metalloproteinases (TIMP)-1 including extravillous in other gynecological tissues, cytotrophoblasts and decidual stromal cells [60–62]. These proteolytic enzymes are involved in the degrada- tion and remodeling of extracellular matrix, which has been implicated in the multistep process of metastasis formation. By activating MMP-2 and MMP-9 promot- expression, GnRH agonists ers

to increase gene

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EGF

GnRHII

GnRH-II receptor ?

EGFR

GnRH-I receptor

A

P

PTP

G α α i

D

B

C

P Sos

P Shc

PKC

JNK

p38

MEK

ERK1/2

AP-1

Migration/ invasion

c-fos expression

Elk-1

Apoptosis

Proliferation

Fig. 2. GnRH-II signaling in ovarian cancer cells. (A) Similar to GnRH-I, GnRH-II acti- vates PTP to inhibit EGFinduced ERK activa- tion, c-fos expression and proliferation. (B) Through PKC, GnRH-II activates ERK and Elk-1 to suppress proliferation. (C) GnRH-II activates JNK to induce migration ⁄ invasion. (D) GnRH-II activates Gai or p38 and AP-1 to induce apoptosis. Dashed arrows represent the EGF-stimulated mitogenic signaling pathway.

[76]. Thus,

Upon stimulation, Ga dissociates from the Gbc dimer and changes to its active GTP-bound form. According to the subtype of their a-subunits, G-proteins can be categorized into four groups: Gs, Gi, Gq ⁄ 11, and G12 ⁄ 13. Gas and Gai mainly exert their effects via stim- ulating or inhibiting, respectively, adenylate cyclase to modulate the production of cAMP. Gaq activates membrane-associated phospholipase C (PLC), which hydrolyzes phosphoinositides to generate the second messengers inositol 1,4,5-triphosphate and diacylglyc- erol, resulting in intracellular Ca2+ mobilization and protein kinase C (PKC) activation. Moreover, Gaq- activated PKC can activate MAPKs, including JNK, ERK and p38 MAPK [68,69]. It is well established that the GnRH receptor interacts with multiple G-pro- teins, and that speciﬁcity is cell context dependent [68]. In hypothalamic neurons, the GnRH receptor interacts with Gaq, Gas and Gai [70]. In pituitary gonadotropes, GnRH preferentially or exclusively stimulates Gaq [71]. However, Gai has been shown to mediate GnRH receptor signaling in tumor cells such as ovarian cancer [50,72,73], endometrial cancer [72,73] and pros- tate cancer [74] cells. Consequently, the downstream signaling pathways and the physiological outcomes of GnRH action may be quite different in the gonado- tropes and extrapituitary tissues.

The mechanism that leads to the inconsistency of GnRH signaling and biological outcome in different tissues is unknown. It is unlikely that divergent signal- ing is due to alternatively spliced variants or mutant

receptors in cancer cells, as expression of the wild-type GnRH receptor has been conﬁrmed in OVCAR-3, lines and EFO-21 and EFO-27 ovarian cancer cell human OSE cells [32,72]. On the other hand, the receptor may oscillate, in a cell context-dependent manner, between multiple conformations, each with signaling complex speciﬁc ligand and intracellular selectivity. According to this hypothesis, the receptor can adopt a conformation that preferentially binds cer- tain ligand(s), in response to different ligand concen- trations. The intracellular signaling complex could in turn stabilize the receptor conformation and favor the binding of the ligand [75]. There is direct evidence sup- porting the existence of multiple conformations of a the GnRH G-protein-coupled receptor receptor in gonadotropes may preferentially bind GnRH-I and be coupled to the Gaq–PLC pathway in order to modulate gonadotropin expression and secre- tion, whereas in cancer cells, the GnRH receptor cou- pling to Gai is selectively recognized and activated by GnRH-II in order to regulate cell proliferation and apoptosis. This kind of conformational preference may be a result of cell context, including cell type and prior exposure to other hormones [75]. Concrete evidence for this speciﬁcity has been generated for the Xenopus GnRH receptor: activation of PKC, which phosphory- lates the C-terminus of the receptor, led to a marked increase in GnRH-II binding to the Xenopus GnRH-I receptor, but had no effect on GnRH-I binding [75]. theory could resolve questions regarding the This

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actions in pituitary gonadotropes and tumor cells remain to be determined.

including why a GnRH system in different tissues, lower binding afﬁnity of GnRH agonists has been observed in tumor cells than in pituitary gonadotropes [75], why GnRH antagonists act like agonists in cancer cells (Table 2), and why GnRH-II is observed to be more potent than GnRH-I in inhibiting cancer cell proliferation through Gai, but less potent in stimulat- ing Gaq-mediated gonadotropin secretion in pituitary gonadotropes [37,77].

tumor

receptor-positive

[27,30,78]. By contrast,

reports

that growth of GnRH-II

Gai and ⁄ or Gaq have been detected in GnRH-I receptor-expressing ovarian cancer cell lines and surgi- cally removed ovarian carcinomas [72,73]. GnRH-I receptor has been shown, by disuccinimidyl suberate cross-linking experiments, to interact physically with Gai and Gaq [72]. Functionally, it has been suggested that GnRH-I receptor couples with Gai, which is com- mon in tumor cells [50,72–74]. Gai is pertussis toxin (PTX)-sensitive and is not affected by cholera toxin. PTX induced ADP-ribosylation of the a-subunit in the cell membrane GnRH-I [50,72–74] and thus impaired GnRH-I receptor-linked cellular events, including GnRH-induced phosphatase activity [50,72,73], apoptosis [50], and antiproliferative incubation with GnRH actions [72,74]. Conversely, agonists substantially antagonized the PTX-catalyzed ADP-ribosylation of Gai [72–74]. Furthermore, Gai sig- niﬁcantly counteracted the forskolin-induced increase in intracellular cAMP levels [74] (Fig. 1G). These ﬁnd- ings strongly indicate that Gai is the major G-protein mediating GnRH actions in tumor cells. In addition to the PTX-sensitive Gai, G-protein bc-subunits also mediated GnRH agonist-induced antiproliferative effects and ERK activation in ovarian cancer cell lines, and such ERK activation was blocked by ectopic expression of the C-terminus of a-adrenergic receptor kinase I, an antagonist of G-protein beta gamma- subunits [81] (Fig. 1B).

lines was

antiproliferative

the

in GnRH actions

In pituitary gonadotropes, PKC and PLC act down- stream of Gaq to relay the GnRH receptor signals. However, the roles of PKC and PLC in GnRH recep- tor signal transduction in tumor cells are less clear. There is evidence that the signaling pathways induced by GnRH-I in pituitary gonadotropes, including PLC and PKC, are not activated by the GnRH-I agonist triptorelin in ovarian, endometrial and breast cancer cell lines [35,36]. Similarly, GnRH-I-stimulated MAPK activation in pituitary aT3 cells was abolished by 4b-phorbol 12-myristate 13-acetate pretreatment (to deplete PKC) or by depletion of Ca2+, whereas the GnRH-I agonist activated MAPK via a PKC-indepen- dent mechanism to inhibit growth of CaOV-3 ovarian cancer cells [81]. However, evidence supporting PKC involvement in tumor cells or extrapituitary tissues is available. Cetrorelix stimulated PKC activity in DU-145 prostate cancer cells, resulting in an increase in phosphorylation of the PKC substrate MARCKS [82]. By activating PKC, GnRH analogs inhibited the EGF receptor (EGFR) signal and the growth of prostate cancer xenografts in athymic mice [83] and cell invasion in vitro [82]. Essentially, prostate

At present, the identity of the GnRH receptor(s) that mediate the antiproliferative actions of GnRH in tumor cells and the agonistic effects of GnRH antago- nists in cancer cells is still controversial. Grundker and co-workers have compared the GnRH responsiveness of SKOV-3 and EFO-27 ovarian cancer cells, and pro- posed that their responsiveness correlates with the expression of GnRH receptors. Accordingly, EFO-27 cells expressing GnRH-I receptor but not GnRH-II receptor responded to the GnRH-I agonist triptorelin but not to antagonists, even at high concentrations (10)5 m) in SKOV-3 cells, which are reportedly GnRH-I receptor-negative but GnRH-II receptor-positive, both the GnRH-I antago- nist cetrorelix and GnRH-II, but not triptorelin, inhib- growth [27,37] or EGF-induced c-fos ited cell lines expressing both GnRH-I expression [78]. Cell and GnRH-II receptors were responsive to all of the treatments. These ﬁndings were in accordance with other receptor- expressing Ishikawa and Hec-IA endometrial cancer cell inhibited by a GnRH-I antagonist [27,38]. Moreover, the GnRH-I agonist-induced anti- proliferative effect was abolished by GnRH-I receptor effects knock-out, whereas induced by the antagonist cetrorelix or GnRH-II agonist persisted [27,79]. These results support the hypothesis that the GnRH-I receptor mediates the actions of GnRH agonists, whereas GnRH antagonists and GnRH-II may act through an additional receptor, i.e. the putative GnRH-II receptor. Although a func- tional GnRH-II receptor has not yet been identiﬁed in humans, the presence and ⁄ or functionality of a type II receptor has not been ruled out. The human GnRH-I antagonist 135-18 and GnRH-II are capable of acti- vating the marmoset GnRH-II receptor, which is over- expressed in COS-7 cells [80]. However, the presence of only one class of speciﬁc, high-afﬁnity ⁄ low-capacity receptors in humans has not been sufﬁciently demon- strated. Binding of the agonist [D-Trp6]GnRH was displaced by the antagonist SB-75, suggesting that both analogs bind to the same receptor on OV-1063 cells [29]. Thus, the precise mechanisms underlying the signaling pathways and biological divergence of

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triptorelin

demonstrated

in a manner that was protein phosphorylation [91], independent of PLC and PKC [92]. The same group further treatment that increased activator protein-1 (AP-1) activity and JunD–DNA binding, and extended the cell cycle [43] (Fig. 1D). As JNK and c-jun are implicated in cell cycle regulation [93], it is logical to hypothesize that the JNK–c-jun–AP-1 pathway mediates the GnRH- induced antiproliferative effect. This pathway may act in concert with NF-jB, as it was shown to protect tumor cells from doxorubicin-induced apoptosis in the same system. JunD is proposed to act as a modulator of cell proliferation and to cooperate with the anti- apoptotic and antiproliferative functions of GnRH. However, further investigation is necessary to resolve the observations by others that the GnRH-I agonist leuprolide and a GnRH-II agonist could not activate JNK [42,81], and that p38 was implicated in GnRH-II- induced ovarian cancer cell apoptosis [57] (Fig. 2D).

cancer cells carrying a mutated EGFR that lacks the target site for PKC are resistant to GnRH-induced in vivo and in vitro growth inhibition [82,83]. Our labo- ratory has recently demonstrated that 12-O-tetradeca- noyl phorbol-13-acetate (a PKC-activating phorbol ester) can mimic the effects of GnRH-I and GnRH-II in stimulating ERK1 ⁄ 2 phosphorylation and antipro- liferation in ovarian cancer cells. Furthermore, the effects of GnRH were abolished by pretreatment with the PKC inhibitor GF109203X [41] (Figs 1C and 2B). By analogy, the PKC inhibitor calphostin C and the activator 12-O-tetradecanoyl phorbol-13-acetate can the antiproliferative block and mimic, respectively, action of the GnRH agonist buserelin on surgically removed uterine leiomyoma [84] cells. In human gran- ulosa-luteal cells, a GnRH agonist stimulates MAPK activation through a PKC-dependent pathway [85]. However, the direct effect on PKC activation and the identity of the PKC isoform activated by GnRH are still under investigation.

to their cognate receptor

and

adaptor

effector molecules

pancreatic

human

cells;

In pituitary gonadotrope cells, there is ample evidence including that GnRH-I activates MAPK members, ERK, JNK and p38, in a PKC-dependent pathway to control gonadotropin secretion [68]. MAPK members mediate distinct roles in GnRH-induced gonadotropin subunit gene transcription [86–88]. In sharp contrast to the situation with pituitary gonadotropes, our under- standing of MAPK activation by GnRH analogs in can- cer cells is rather limited. In ovarian cancer OVCAR-3 cells and placental cancer JEG-3 cells, we have demon- strated biphasic activation of ERK by [D-Ala6]GnRH. High doses of GnRH agonist (10)7 and 10)6 m) signiﬁ- cantly activated ERK, whereas a low dose (10)10 m) resulted in decreased ERK activation [89]. In another study, phosphorylation of ERK, Sos and Shc was induced by the GnRH-I agonist leuprolide [81]. Leupro- lide-induced ERK activation was rapid (within 5 min) and long-lasting (sustained up to 24 h). ERK appeared to mediate the antiproliferative action of GnRH and such growth inhibition could be reversed by the mito- gen-activated protein kinase kinase (MEK) inhibitor PD98059 [81]. The dual roles of ERK in mediating mitogenic effects (by growth factors) and antiprolifera- tive effects (by GnRH) seem contradictory. Paradoxical actions of ERK have also been reported in PC-12 cells: transient activation induced by EGF led to prolifera- tion, and nerve growth factor-induced prolonged ERK activation caused differentiation and cessation of prolif- eration [90]. It has been suggested that the duration of ERK activation is important in determining its actions and thus the resultant cell fate [90].

In addition to ERK, JNK was activated by triptore- lin through induction of c-jun mRNA expression and

In addition to the direct antiproliferative signal elic- ited through PKC-dependent or PKC-independent pathways, the antiproliferative effect on tumor cells of GnRH may arise from its ability to activate phospha- tases and counteract the mitogenic signals induced by growth factors [94] (Fig. 1A). In OSE and epithelial ovarian carcinomas, EGF and various growth factors are secreted and function locally to promote tumor proliferation and progression [95]. Binding of growth factors tyrosine kinases induces receptor dimerization and autophosphoryla- tion. Phosphorylated receptor tyrosine kinases phos- phorylate to subsequently initiate a phosphorylation cascade that is important for the growth-promoting and tumorigenic functions of growth factors. Propagation of the phos- phorylation cascade and its physiological effects can be terminated by protein phosphatases. It has been shown that EGFR, when stimulated by EGF, may phosphor- ylate itself and other cellular substrates, including Src, in cotreatment with [D-Trp6]GnRH reversed the effect of EGF and led to the dephosphorylation of these proteins [96]. In the tumor cells, phosphotyrosine plasma membrane of phosphatase (PTP) or serine ⁄ threonine protein phos- phatase 2A (PP2A) were shown to be activated by GnRH agonists suggesting that [50,54,72,97–100], GnRH-I increased the turnover rate of protein phos- phorylation ⁄ dephosphorylation and that EGFR is a target of the dephosphorylation activity [72,96]. As a result, EGFR phosphorylation [72] and the down- stream signaling and mitogenic effects of EGF were abrogated, including EGF-induced MAPK activation immediate early gene c-fos expression [78] and [94],

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EGF-induced

Three trials have been completed that compared the use of platinum-based chemotherapy alone or in com- bination with a GnRH agonist as ﬁrst-line therapy for ovarian cancer [121–123]. A prospective randomized double-blind trial enrolled 135 patients with stage III or IV epithelial ovarian carcinoma, and showed that suppression of endogenous gonadotropins by conven- tional doses of the GnRH agonist triptorelin produces no relevant beneﬁcial effects in patients with advanced ovarian carcinoma who receive standard surgical cyto- reduction and standard platinum-based chemotherapy [121]. In the other two studies, patients received carbo- platin-containing polychemotherapy and cisplatin alone or chemotherapy plus triptorelin, but no signiﬁcant dif- ferences were seen in terms of response, survival and time to progression [122,123]. The ineffectiveness of the GnRH agonist in combination with chemotherapy is postulated to be due to the neutralization of its direct antiproliferative effects by its antiapoptotic activity, as demonstrated by the in vitro data [53,54,124].

proliferation [92]. Downregulation of receptors for EGF and ⁄ or insulin-like growth factor-I (IGF-I) by GnRH antagonist [101] and in ovarian cancer-xeno- grafted nude mice have been reported [31,102]. Inter- ference of growth factor signaling by GnRH analogs has also been demonstrated in prostate cancer cells. GnRH-I analogs abrogated the mitogenic effects of EGF and IGF-I and inhibited prostate cancer growth their receptors [103,104] by reducing expression of [103–105], as well as by inhibiting EGF- and IGF-I- induced receptor phosphorylation [103,104] and c-fos expression. GnRH-II agonist was reported to act in a similar fashion, i.e. enhancing PTP activity and thus reducing EGFR phosphorylation, MAPK activation and c-fos expression [79] (Fig. 2A). Moreover, GnRH-activated phosphatase activity has also been implicated in its antiapoptotic function. Doxorubicin decreased the activity of a crucial phosphatase in apoptosis control (PP2A), and induced ovarian cancer cell apoptosis. Cotreatment with the GnRH-I agonist leuprolide partially restored PP2A activity and antagonized doxorubicin-induced apopto- sis [54] (Fig. 1E).

Clinical studies on GnRH agonists and antagonists in ovarian cancer

In vitro data demonstrated that antagonists provided a greater inhibitory effect on ovarian cancer prolifera- tion than agonists [29]. Clinically, as GnRH-I antago- nists do not possess intrinsic gonadotropic activity, the initial ‘ﬂare-up’ phenomenon, which is common in agonist treatment, can be avoided. This makes antago- nists better tolerated and capable of blocking gonado- tropin secretion within a short time frame [125]. A clinical trial of the GnRH antagonist cetrorelix was the patients had conducted on 17 patients. All of relapsed disease after standard chemotherapy before entering into the trial. Three patients (18%) experi- enced a partial remission with cetrorelix treatment that lasted 2, 6 and 7 months, and six women (35%) had disease stabilization for 1–12 months. The median survival was 17 months [126].

triptorelin. No objective

Conclusions and future prospects

for patients with disease

receptor and a role of

[106–120]. The majority of

these

To date, our understanding of the GnRH system in tumor cells is still far from complete, especially with regard to the newly identiﬁed GnRH-II isoform and the ‘putative’ GnRH-II receptor. Although there are suggestive data supporting the existence of a functional mammalian GnRH-II the GnRH-II receptor in mediating the antiproliferative effects of GnRH-I antagonists and GnRH-II, direct evidence for a functional human GnRH-II receptor and the details of its downstream signaling mechanism are certainly of great physiological importance and research interest. The superior antiproliferative effects of GnRH-II as compared to GnRH-I make GnRH-II an attractive target for investigation, and the hormonal

In a limited number of studies, GnRH-I agonists have been evaluated for their potential as second-line ther- apy in patients with refractory and recurrent ovarian cancer who had failed at least one chemotherapy regi- men. In 2001, the European Organization of Research and Treatment of Cancer Gynecological Cancer Coop- erative Group completed the largest reported series of GnRH agonist trials. Seventy-four patients with pro- gressive ovarian cancer who had previously undergone platinum-based therapy were treated with the GnRH responders were agonist observed. Eleven of 68 evaluable patients (16%) had stable disease. The median progression-free survival was 5 months in patients with disease stabilization and 2 months for all evaluable patients. The median sur- stabilization was vival 17 months, whereas for all patients it was 4 months. This study showed that treatment with the GnRH agonist triptorelin has only modest efﬁcacy in patients chemotherapy pretreated with platinum-containing [106]. Table 2 summarizes 15 clinical trials, beginning as early as 1988, that have used three different GnRH agonists (leuprolide acetate, triptorelin, goserelin) on relapsed platinum-resistant ovarian cancer patients (Table 2) trials involved only a limited number of patients.

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10 Shoham Z (1994) Epidemiology, etiology, and fertility drugs in ovarian epithelial carcinoma: where are we today? Fertil Steril 62, 433–448.

11 Rao BR & Slotman BJ (1991) Endocrine factors in

such as

common epithelial ovarian cancer. Endocr Rev 12, 14– 26.

12 Parrott JA, Doraiswamy V, Kim G, Mosher R & Skin- ner MK (2001) Expression and actions of both the fol- licle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer. Mol Cell Endocrinol 172, 213–222.

regulation of GnRH-II expression in ovarian cancer cells is presently under investigation in our laboratory. The widespread expression of the GnRH receptor in ovarian carcinomas and the well-documented in vitro antiproliferation and apoptosis, effects, strongly support the candidacy of GnRH as a promis- ing therapeutic approach for ovarian cancer. Elucida- tion of the efﬁcacy and modes of actions of GnRH-I and GnRH-II, as well as their interactions with growth factors that are known to be important in ovarian cancer progression, is undoubtedly warranted.

Acknowledgements

13 Syed V, Ulinski G, Mok SC, Yiu GK & Ho SM (2001) Expression of gonadotropin receptor and growth responses to key reproductive hormones in normal and malignant human ovarian surface epithelial cells. Cancer Res 61, 6768–6776.

14 Choi KC, Kang SK, Tai CJ, Auersperg N & Leung

P.C.K.L. is the recipient of a Child & Family Research Institute Distinguished Scholar Award. W.K.S., J.C.C. and S.L.P. were recipients of graduate studentship awards from The Interdisciplinary Women’s Repro- ductive Health Research Training Program.

PC (2002) Follicle-stimulating hormone activates mito- gen-activated protein kinase in preneoplastic and neo- plastic ovarian surface epithelial cells. J Clin Endocrinol Metab 87, 2245–2253.

15 Choi JH, Choi KC, Auersperg N & Leung PC (2005)

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