Báo cáo khoa học: Gonadotropin-releasing hormone: GnRH receptor signaling in extrapituitary tissues docx

MINIREVIEW Gonadotropin-releasing hormone: GnRH receptor signaling in extrapituitary tissues Lydia W T Cheung and Alice S T Wong School of Biological Sciences, University of Hong Kong, China Keywords cross-talk; extrapituitary; GnRH; GnRH receptor; MAPK; metastasis; pituitary; receptor tyrosine kinase; signaling; tumor Correspondence A S T Wong, School of Biological Sciences, University of Hong Kong, 4S-14 Kadoorie Biological Sciences Building, Pokfulam Road, Hong Kong, China Fax: +852 2559 9114 Tel: +852 2299 0865 E-mail: awong1@hku.hk (Received 14 April 2008, revised 28 May 2008, accepted 11 June 2008) doi:10.1111/j.1742-4658.2008.06677.x Gonadotropin-releasing hormone (GnRH) has historically been known as a pituitary hormone; however, in the past few years, interest has been raised in locally produced, extrapituitary GnRH GnRH receptor (GnRHR) was found to be expressed in normal human reproductive tissues (e.g breast, endometrium, ovary, and prostate) and tumors derived from these tissues Numerous studies have provided evidence for a role of GnRH in cell proliferation More recently, we and others have reported a novel role for GnRH in other aspects of tumor progression, such as metastasis and angiogenesis The multiple actions of GnRH could be linked to the divergence of signaling pathways that are activated by GnRHR Recent observations also demonstrate cross-talk between GnRHR and growth factor receptors Intriguingly, the classical Gaq–11-phospholipase C signal transduction pathway, known to function in pituitary gonadotropes, is not involved in GnRH actions at nonpituitary targets Herein, we review the key findings on the role of GnRH in the control of tumor growth, progression, and dissemination The emerging role of GnRHR in actin cytoskeleton remodeling (small Rho GTPases), expression and ⁄ or activity of adhesion molecules (integrins), proteolytic enzymes (matrix metalloproteinases) and angiogenic factors is explored The signal transduction mechanisms of GnRHR in mediating these activities is described Finally, we discuss how a common GnRHR may mediate different, even opposite, responses to GnRH in the same tissue ⁄ cell type and whether an additional receptor(s) for GnRH exists Introduction The hypothalamic gonadotropin-releasing hormone (GnRH) is a decapeptide that plays a critical role in the regulation of reproduction GnRH-I (pGlu-HisTrp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) is the first GnRH isoform discovered in mammalian brain Its major role is to stimulate pituitary secretion of gonadotropins, luteinizing hormone and follicle-stimulating hormone, which in turn stimulate the gonads for steroid production Subsequently, a second isoform of GnRH (His5, Trp7, Tyr8) (GnRH-II) has been isolated from chicken brain It is also highly conserved among vertebrates, including mammals [1] However, in contrast to GnRH-I, GnRH-II is expressed at significantly higher levels outside the Abbreviations EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-related kinase; FAK, focal adhesion kinase; FGF, fibroblast growth factor; GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NF-jB, nuclear factor kappa B; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; Pyk2, proline-rich tyrosine kinase 2; RTK, receptor tyrosine kinase; uPA, urokinase-type plasminogen activator; VEGF, vascular endothelial growth factor FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5479 GnRH receptor signaling L W T Cheung and A S T Wong brain and is particularly abundant in the kidney, bone marrow, and prostate [2] This leads to the speculation that GnRH-II may have distinct physiological functions from those of GnRH-I In line with this is the observation that although GnRH-II can stimulate gonadotropin secretion, its efficiency is much lower than that of GnRH-I (only about 2% of that of GnRH-I) [3] This suggests that the primary role of GnRH-II is not in the regulation of gonadotropin secretion Instead, this peptide has been shown to act as a neuromodulator [4] The exact actions of GnRH-II in peripheral tissues are not entirely understood, but this is certainly an important topic for investigation which may offer an opportunity to elucidate the undisclosed complexity of GnRH In this minireview, we will focus on recent progress in understanding the roles of GnRH-I and GnRH-II in extrapituitary tissues, in particular its emerging role in tumor growth, invasion, and metastasis We will also describe the molecular mechanisms underlying these effects, focusing on the roles of proteolysis, adhesion, and signaling, as well as our still-emerging understanding of receptor cross-talk with other pathways Finally, we will discuss two important outstanding questions in the field regarding what might distinguish the different responses to the same ligand (GnRH) and whether an additional receptor(s) for GnRH exists in humans Localization of GnRH receptor (GnRHR) in peripheral reproductive tissues The initial interest in extrapituitary GnRHR stemmed primarily from observations in the 1980s that GnRH analogs can inhibit the growth of nonpituitary tumor cell lines [5] Soon after this, a functional type I GnRHR was demonstrated in a variety of normal human reproductive tissues (e.g breast, endometrium, ovary, and prostate) and tumors derived from these tissues In the ovary, GnRHR mRNAs are expressed in granulosa-luteal cells, and increased expression of GnRHR correlates with follicular growth and development [6] GnRHR binding has been demonstrated in luteinized granulosa cells, late follicles and developing corpora lutea, but not in primordial, early antral and preovulatory follicles [7,8] This stage-specific expression of GnRHR in the human granulosa and luteal cells suggests a role for GnRH in the regulation of ovarian physiology, particularly ovulation, follicular atresia and luteolysis The presence of GnRHR protein and mRNA has also been demonstrated in human ovarian tumor specimens, ovarian cancer cell lines and 5480 their tissue of origin, ovarian surface epithelium [9,10] Interestingly, levels of GnRHR seem to be associated with cancer grading and have been reported to be elevated in advanced stage (stages III and IV) as compared to early stage (stages I and II) ovarian carcinomas [11] Our recent findings that GnRH can promote the motility and invasiveness of ovarian cancer cells further corroborate the view that GnRH may play a crucial role in tumor progression ⁄ metastasis [12,13], and these findings will be discussed in a later section Using [125I][d-Trp6]GnRH, specific receptor binding has been detected in membranes from 24 of 31 (77%) endometrial carcinomas and from three of 13 (23.1%) nonmalignant human endometrial specimens [14] GnRHR mRNA has been clearly detected in surgical endometrial carcinoma specimens and endometrial carcinoma cell lines [15,16] As with normal myometrium, most benign neoplasms studied thus far, including uterine leiomyoma, also possess GnRHR [17] Early studies showed that the human placenta contains specific binding sites for GnRH that interact with GnRH agonists and antagonists [18] Later on, GnRHR was localized to the cytotrophoblast and syncytiotrophoblast cell layers [19,20] Temporal expression of GnRHR in the placental cells at different weeks of gestation has been observed, in parallel with the time-course of chorionic gonadotropin secretion during pregnancy [21], suggesting that the expression of the receptor is a function of pregnancy stage The presence of GnRHR has been demonstrated in numerous human breast cancer cell lines and tumor biopsy specimens [22–24] GnRHR was immunolocalized in the cytoplasm in 37 of 58 (64%) invasive ductal carcinoma cases [23] The expression of GnRHR in normal human breast tissue is still controversial, but the sample size may have been too small to allow any definite conclusion [22,25] GnRHR is also present in prostate cancer cells, as shown by radioligand-binding studies, PCR, and western blotting analysis [26,27] GnRHR immunoreactivity is localized to the luminal and basal epithelial cells in benign and malignant prostate tissues In this study, the relative GnRHR mRNA levels showed a wide range of individual differences that were unrelated to the histological grades of the 16 cases [27] There does, however, appear to be significantly higher expression of GnRHR in hormone-refractory prostate carcinoma than in other types of prostate tumor (n = 80) [28] Although these extrapituitary GnRHRs share the same cDNA nucleotide sequence and encode transcripts and proteins of the same size as the pituitary FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS L W T Cheung and A S T Wong GnRHR [20,26,29], they also differ in several ways First, cell surface receptor expression in extrapituitary sites is low as compared to that of the pituitary [15,27] This may underlie the greater effect of the GnRHR ligands on the gonadotropes Second, there are at least two classes of GnRHR: one has high affinity [with nanomolar dissociation constants (Kd)] for GnRH, and one has low affinity (with micromolar Kd values) for GnRH The high-affinity GnRH-binding sites are commonly regarded as being the same as the GnRHR of the pituitary gland Whereas in most of the reported cases, both the low-affinity and high-affinity GnRHR have been found in extrapituitary tissues [30–33], in some cases, only low-affinity GnRHR could be detected [10,18,34], and in others, e.g in endometrial cancers and nonmalignant endometrial specimens, only the high-affinity GnRHR has been demonstrated [14] The exact role of each of these receptors and the implications of differential levels of expression remain to be elucidated Functions of GnRH-I and GnRH-II in cancers Tumor growth Over the last two decades, both GnRH agonists and antagonists have been widely used as therapeutics in treating sex steroid-dependent tumors The majority of these GnRH analogs, when given continuously, inhibit gonadotropin synthesis and secretion via downregulation of the pituitary GnRHRs This indirect mechanism of action has provided the rationale for the use of GnRH analogs in the treatment of hormone-dependent tumors for many years Only since the detection of GnRHR in extrapituitary tissues has there been increasing interest in its direct action on tumor cells GnRH-I analogs have direct antiproliferative effects on ovarian cancer cells, which is linked to the disruption of the cell cycle at G0 ⁄ G1 [31,35,36] On the other hand, several independent in vitro studies failed to demonstrate significant growth inhibition by GnRH-I agonists, even at fairly high concentrations (micromolar range) [37,38] In fact, a biphasic impact of GnRH-I agonists on growth has been reported: whereas GnRH-I agonists at high dose (1 lm) inhibit cell proliferation in vitro, cells treated with agonists at low dose (10 nm) show significant growth stimulation [39] Further studies demonstrated that nanomolar concentrations of GnRH-I agonists also increase cell survival under multiple stress conditions, including DNA replicationspecific cytotoxic agents and UV radiation [40] GnRH receptor signaling GnRH-II has antiproliferative effects on ovarian cancer cells [41–43] Although it has been suggested that this effect of GnRH-II is mediated through the type I GnRHR [43], there are other findings implicating a type I GnRHR-independent action [41,42] It is interesting to note that although both GnRH-I agonists and antagonists exert antiproliferative effects, the effects of GnRH-I antagonists are stronger than those of the agonists [44] This difference has also been seen in an in vivo model, which demonstrates a significant inhibition of tumor growth by GnRH-I antagonists but not GnRH-I agonists [45] The advantage of GnRH antagonists over the agonistic peptides is probably due to the fact that they inhibit the secretion of gonadotropins and reduce sex steroid levels immediately after application, thus achieving rapid therapeutic effects, whereas repeated exposure to agonistic agents is required to induce functional desensitization of the gonadotropes [46] Treatment of human endometrial cancer cells (cell line Ishikawa) with the GnRH-I antagonist SB-75 results in growth inhibition, mainly due to the Fas ⁄ Fas ligand-mediated apoptotic pathway, whereas GnRH-I agonists have no effect on the same cell line [15,47,48] Another endometrial carcinoma cell line, HEC-1A, also exhibits differential responses to different GnRH agonists and antagonists [15,30,36,48] GnRH-II has been shown to have antiproliferative effects on endometrial carcinoma cells [41] The effects of GnRH-I are abrogated after type I GnRHR knockout [36], whereas those of the GnRH-I antagonist cetrorelix and of GnRH-II persist [41] These findings suggest that the antiproliferative effects of cetrorelix and GnRH-II are not mediated through the type I GnRHR GnRH-I has been demonstrated to have antiproliferative effects on prostate cancer cells [49–51], except in one in vivo study [52] This antiproliferative effect appears to be independent of the androgen receptor status of the prostate carcinoma cells, as both androgen-sensitive LNCaP cells and androgen-resistant DU-145 cells remain sensitive to GnRH [49,50] Activation of GnRHR may mediate these effects via direct induction of apoptosis through caspase activation [53] Compatible with a role for GnRH in survival at low doses, an enhancing effect of GnRH was observed when cells were treated with a low concentration (100 pm) of GnRH-I agonist [54] GnRH-II was shown to have an antiproliferative effect on DU-145 cells and growth-stimulatory effect on TSU-Pr1 cells, but the type I GnRHR was not involved [55] The influence of GnRH on the growth of human breast cancer cells was first studied with MCF-7 cells [56], and both in vitro and in vivo proliferation of FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5481 GnRH receptor signaling L W T Cheung and A S T Wong breast cancer cells could be inhibited by both agonistic and antagonistic analogs of GnRH [57,58] However, higher efficiency of GnRH antagonists in growth inhibition than that of GnRH agonists has been reported [24,58] Invasion and metastasis The observation that GnRH controls tumor growth suggests a regulatory role for this peptide in the metastatic behavior of cancer cells This hypothesis is supported by studies showing that GnRH-I and GnRH-II can affect the expression of several extracellular matrix-degrading enzymes in human extravillous cytotrophoblasts and decidual stromal cells to facilitate implantation [59,60] However, its potential role in cancer metastasis has just begun to be revealed Metastasis is a complex phenomenon that requires several specific steps, such as decreased adhesion, increased motility, and proteolysis The effects of GnRH in tumor metastasis are mediated through the regulation of adhesion molecules, Rho GTPases, and two families of metastasis-related proteinases, the matrix metalloproteinases (MMPs) and the urokinase-type plasminogen activator (uPA) system, at several levels: mRNA transcription, secretion, and proenzyme activation The ability of GnRH to regulate metastasis was first reported in melanoma cells [61] High doses of GnRH-I analog, at micromolar concentrations, significantly reduces the ability of melanoma cells to invade and migrate [61] Preliminary data (R M Moretti, M Monagnani Marelli, J C van Groeninghen, M Motta & P Limonta, unpublished results, 2003) indicate that this inhibitory action is due to the effects of integrins and MMPs [62] We were the first to report possible metastatic activity of GnRH-I in tumors of the female reproductive tract [12] GnRH-I exerts a biphasic effect on cellular migration and invasion: whereas lower (nanomolar) concentrations of the GnRH-I agonist stimulate cellular migration and invasion in a dose-dependent manner, high (micromolar) concentrations are not as efficient This proinvasive effect is mediated through activation of metastasis-related proteinases, in particular MMP-2 and MMP-9 [12] Moreover, GnRH-I is able to transactivate the MMP-2 and MMP-9 promoters, which means that GnRH can be considered to be a new member of MMP-2 and MMP-9 transcriptional modulators Like GnRH-I, native GnRH-II and its synthetic analog also induce a similar biphasic regulation of ovarian cancer invasion [13] The finding that small interfering RNA-mediated downregulation of type I GnRHR completely reversed the effects of both 5482 GnRH-I and GnRH-II on cell invasion supports the view that the same receptor, type I GnRHR, is essential for the effects of GnRH-I and GnRH-II in ovarian cancer cells The decrease in uPA activity of cytosol from Dunning R3327H rat prostate tumors after treatment with GnRH-I analogs suggests that GnRH may be an important factor in reducing the invasiveness of prostate cancer [63] High doses of GnRH-I agonists and antagonists have been reported to attenuate the invading capacity of both androgen-dependent and androgen-independent prostate cancer cells by modulating E-cadherin-mediated cell–cell contacts and production of uPA and its inhibitor (plasminogen activator inhibitor-1) [64–66] GnRH has also been shown to regulate cell motility through its interaction with the small GTPases Rac1, Cdc42, and RhoA, which are involved in the regulation of actin polymerization [67] Up to now, there has been only one study, by Von Alten et al., investigating the role of GnRH in breast cancer metastasis, using a coculture system with human osteosarcoma cells to analyze tumor cell invasion to bone [68] The consequences of GnRHR activation are complex and appear to be cell context dependent: whereas treatment of cells with the GnRH-I agonist triptorelin, the GnRH-II agonist [d-Lys6]GnRH-II and the GnRH-I antagonist cetrorelix decreases the invasion rate in most breast cancer cell lines, these agents have no significant effect in the GnRHR-positive MDA-MB-435 cells [68] Further investigations are required to elucidate the reason why the MDA-MB-435 cell line reacts differently Organ-specific homing and colonization of cancer cells are important and interesting features of metastasis A role for GnRH has also been suggested in the regulation of the immune response and metastasis GnRH-I and GnRH-II are expressed in human normal and cancerous T-cells GnRH triggers laminin receptor gene expression, adhesion to laminin, in vitro chemotaxis, and in vivo homing to specific organs [69] Angiogenesis Angiogenesis is crucial to a number of physiological and pathological processes, such as reproduction, development, and tissue repair, as well as tumor growth and metastasis Vascular endothelial growth factor (VEGF) is implicated as the most important angiogenesis inducer, because of its potency in a variety of normal and tumor cells Other angiogenic factors include fibroblast growth factor (FGF), platelet-derived growth factor and the angiopoietin family FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS L W T Cheung and A S T Wong The effect of GnRH on angiogenesis in the ovary, in which this neovascularization is necessary for follicular and luteal function, has been demonstrated A recent in vivo study using rats revealed that an application of the GnRH-I agonist leuprolide acetate decreases the protein expression of VEGF and angiopoietin-1 and their receptors in ovarian follicles, and that this can be reversed by coinjection of the GnRH antagonist antide [70] A similar inhibitory effect on angiogenesis can be observed in marmosets injected with the GnRH-I antagonist antarelix [71] However, VEGF mRNA expression is unaffected by the treatment The clinical response of uterine shrinkage after GnRH analog treatment and a pathological role of FGF-2, VEGF and platelet-derived growth factor in uterine leiomyoma growth and vascularization has also been suggested [72] Considering that angiogenesis is an important process in many human cancers, it would be GnRH receptor signaling very interesting to determine whether GnRH also plays a key role in tumor angiogenesis Intracellular signal transduction Upon GnRH binding, GnRHR undergoes a conformational change and stimulates a unique G-protein Interestingly, the classical Gaq–11-phospholipase C signal transduction pathway, which is known to operate in the pituitary, is not involved in the antitumor activity of GnRH analogs Rather, GnRHRs couple to Gai in these tumors and result in the activation of several downstream signaling cascades [73,74], such as mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K), and nuclear factor kappa B (NF-jB) signaling The GnRH-induced signaling pathways in extrapituitary tissues are shown schematically in Fig Fig Schematic representation of GnRHR signaling in extrapituitary tissues Binding of GnRH to GnRHR triggers several intracellular signaling cascades and cross-talk with mitogenic signaling, depending on the cell context Some of these signaling modules can transduce extracellular signals to the nucleus and thereby regulate genes that are involved in cell growth, metastasis, or survival Arr, b-arrestin; CREB, cAMP response element-binding protein; FGFR, fibroblast growth factor receptor; HB-EGF, heparin-binding EGF; IjB, inhibitory factor kappa B; IGFR, IGF receptor; MEK, mitogen-activated protein kinase kinase; MLK3, mixed-lineage kinase 3; PTP, protein tyrosine phosphatase; Sos, son of sevenless; TNF-a, tumor necrosis factor alpha FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5483 GnRH receptor signaling L W T Cheung and A S T Wong MAPK The major MAPK cascades include extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38 MAPK Many studies have shown that the MAPK pathway is critical for GnRH activities, which provides an important link for the transmission of signals from the cell surface to the nucleus Activation of MAPK by GnRH involves distinct upstream pathways in generating tissue-specific and cell-specific signaling (Fig 1) This can occur at different levels via different mechanisms: (a) second messengers [protein kinase C (PKC) and cAMP] [26]; (b) receptor-interacting proteins such as Src and b-zarrestin [53,75]; and (c) upstream kinases such as MAPK ⁄ ERK kinase and PI3K [53,76] For example, GnRH-I induces apoptosis in DU-145 prostate cancer cells via JNK, which is activated through two independent mechanisms [53] Activation of the pathway is dependent on c-Src with concomitant decrease in Akt activity, and the combination of these two events relieves the inhibition of the upstream activator of JNK, MLK3 [53] (Fig 1) Interestingly, although ERK1 ⁄ is phosphorylated through epidermal growth factor receptor (EGFR) under the same conditions, this pathway is not involved in the apoptotic effects These findings demonstrate that activation of the two MAPKs, which lead to distinct physiological outcomes, is separated already at the upstream levels In the ovarian cancer cell line CaOV-3, prolonged stimulation of ERK1 ⁄ through Shc and son of sevenless is required for GnRH-I-mediated growth inhibition [76] Consistent with the fact that sustained activation of ERK1 ⁄ is often correlated with cell cycle progression, GnRH-I-induced growth inhibition is attributed to G1 arrest [76] Moreover, the signaling cascade was shown to be initiated by Gbc, supporting the notion that the post-GnRHR signaling cascade in extrapituitary cells is different from that in pituitary cells GnRH-induced MAPK activation has also been shown in another ovarian cancer cell line, OVCAR-3 Both ERK1 ⁄ and p38 MAPK mediate the antiproliferative effects of GnRH-I and GnRH-II in a PKC-dependent manner [43,77] GnRH-II induces the activation of activator protein-1 transcription factor via p38 MAPK, suggesting a potential role of activator protein-1 in ovarian cancer cell growth [77] The JNK pathway also drives tumor invasion and migration in ovarian cancer cells [12], but the activation mechanism(s) remains to be elucidated Temporal and spatial differences in cellular signaling may have significant phenotypic manifestations [78,79] For example, sustained activation of ERK1 ⁄ has been 5484 implicated in nerve growth factor-mediated neuronal differentiation of PC12 cells, whereas a rapid and transient activation is associated with growth factormediated proliferation of PC12 cells [80] Thus, the duration of kinase activation seems to be a major determinant of signal outcome We have shown differential regulation of ERK1 ⁄ 2, p38 MAPK, and JNK by GnRH-I with sustained signaling through the JNK pathway in ovarian cancer cells [12] Consistently, GnRH-stimulated MMP-2 and MMP-9 expression, secretion and cell invasion were attenuated by specific inhibition of JNK but not of ERK1 ⁄ and p38 MAPK, suggesting that prolonged activation of JNK may contribute to a more invasive phenotype Strong and sustained activation of MAPK has been reported to be necessary for its cytoplasm-to-nucleus translocation, and thereby contributes to the regulation of gene expression [79,81] It will be interesting to see whether sustained activation of JNK leads to its nuclear translocation, which is required for GnRH-stimulated cell invasion The JNK pathway targets multiple transcription factors, including c-Jun, c-Fos, ATF and PEA, and putative binding sites for these DNA-binding proteins are present in the MMP promoters [82] Whether these putative regulatory elements participate in the GnRH-dependent activation of the MMP-2 and MMP-9 genes remains to be determined Cross-talk with mitogenic signaling Cross-talk between cell surface receptors, which has been recognized as a mechanism capable of generating signal diversity, is now receiving further interest Figure illustrates the cross-talk between GnRHR and receptor tyrosine kinases (RTKs) For instance, GnRH causes transactivation of RTKs, such as EGFR [75,78,83] MMP-2 and MMP-9 seem to be essential for GnRH-induced EGFR activation by cleavage of the heparin-binding epidermal growth factor (EGF) precursor [84] Transactivation of EGFR has been shown to activate ERK1 ⁄ 2, as GnRH-induced ERK1 ⁄ phosphorylation can be abolished in the presence of the EGFR inhibitor AG1478 [53,78] However, the biological importance of ERK1 ⁄ activation in response to this cross-talk still remains elusive Negative cross-talk between GnRHR and growth factor receptors has also been described For instance, the antiproliferative effects of GnRH-I and GnRH-II agonists are mediated through attenuation of EGFR signaling in many reproductive tumor cells [57,66,85– 87] In prostate cancer cells, cetrorelix is able to counter EGFR-dependent adhesive signaling through a PKC-dependent mechanism [66] Activation of FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS L W T Cheung and A S T Wong GnRHR appears to mediate these effects via activation of phosphotyrosine phosphatase, thereby reducing EGF-induced EGFR autophosphorylation, resulting in downregulation of mitogenic signal transduction and cell proliferation [85,86,88] A negative regulatory interaction between GnRHR and mitogenic signaling pathways has also been reported in human prostate cancer cells via insulin-like growth factor GnRH-I agonists inhibit expression of the insulin-like growth factor receptor, receptor tyrosine phosphorylation, and the subsequent downstream activation of Akt [89–91] Another example is FGF-2 GnRH analog treatment has been shown to block cell proliferation and invasion induced by FGF-2 stimulation [65] PI3K The PI3K signaling pathway and its downstream target Akt (also named protein kinase B) has been implicated in promoting cell survival, proliferation, and invasion In uterine leiomyomas, the GnRH-I agonist leuprolide acetate causes a significant reduction in PI3K ⁄ Akt activity and inhibits the expression of the antiapoptotic proteins (c-FLIP and PED ⁄ PEA15), thereby inducing apoptosis [92] In the SKOV-3 ovarian cancer cell line, GnRH-I and GnRH-II interfere with activation of the PI3K ⁄ Akt cascade, and this is is associated with the inhibitory effects of GnRH on cell invasion [13] Although PI3K ⁄ Akt and MAPK are two parallel pathways in some cell types, they are two related pathways in the mediation of GnRH actions, as inhibition of PI3K ⁄ Akt can alter the activation of MAPK For instance, in prostate cancer cells, stimulation of PI3K ⁄ Akt releases mixed-lineage kinase 3, which in turn activates the JNK pathway, and this positive regulation is important for the proapoptotic effect of GnRH-I (Fig 1) [53] PI3K ⁄ Akt is also an upstream kinase of ERK1 ⁄ 2, and EGFR transactivation by GnRH-I may be required for the activation of this cascade [75,93] Other signaling pathways Activation of NF-jB is important for the protection against apoptosis in ovarian tumors induced by the GnRH-I agonist tiptorelin [94] The effect is probably mediated by the Gai-coupled GnRHR, and receptor activation causes nuclear translocation of NF-jB [94] Unlike the other signaling pathways studied, the GnRH-I-induced NF-jB activation appears to be independent of the cross-talk between GnRHR and growth factor signaling, as treatment with phosphatase inhibi- GnRH receptor signaling tor has no effect on the activation of NF-jB [94] It has also been shown that GnRH-I suppresses interleukin-8 expression via attenuation of tumor necrosis factor alpha-induced NF-jB signaling in endometriotic stromal cells (Fig 1) [95] These data suggest that modulation of cytokine signal transduction by GnRH may be one of the mechanisms contributing to its growth-inhibitory effect The non-RTKs focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2) are the predominant mediators of integrin signaling GnRHR has been shown to signal through these molecules, suggesting a role for GnRH in cytoskeletal reorganization In human endometrial cancer cells (HEC-1A), b3-integrindependent activation of FAK is associated with the inhibitory effects of GnRH-I and GnRH-II on growth [96] Leiomyoma regression induced by GnRH-I agonists has been suggested to be mediated, at least in part, through a mechanism involving suppression of FAK [97] Maudsley et al demonstrated a novel signaling cascade of GnRHR that functionally antagonizes the actions of testosterone and inhibits prostate tumor growth [98] GnRH controls the tyrosine phosphorylation status of the focal adhesion proteins Pyk2 and Hic-5 This alteration of the focal adhesion dynamics then results in nuclear translocation of the androgen receptor, which renders it transcriptionally inactive [98] Mechanisms underlying the diverse responses to GnRH action As discussed earlier, GnRH and its agonists have a dual and biphasic action: whereas low concentrations (0.1–10 nm) of GnRH stimulate cell proliferation, migration and invasion in a dose-dependent manner, high concentrations (100 nm to lm) inhibit these functions [12,13,39] Moreover, the same dose of GnRH can elicit completely opposite responses in cells derived from the same tissue We demonstrated that in two human ovarian cancer cell lines, OVCAR-3 and SKOV-3, GnRH-I and GnRH-II induce invasion of OVCAR-3 cells, but inhibit the invasiveness of SKOV3 cells [13] A similar difference has been found in the effects of GnRH on cell proliferation and cell migration in the prostate carcinoma cell lines TSU-Pr1 and DU-145 [67] Whereas GnRH-I and GnRH-II stimulated cell proliferation, induced actin cytoskeleton remodeling and promoted migration in TSU-Pr1 cells, they were inhibitory in DU-145 cells [67] The observation that GnRH-I and GnRH-II have no significant effect in cell lines with type I GnRHR depletion indicates that the type I GnRHR is indispensable for the FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5485 GnRH receptor signaling L W T Cheung and A S T Wong Table Potential mechanisms that underlie the diverse responses to GnRH Determinants References Treatment conditions, e.g duration and dose Different Ga subtypes Different G-protein subunits GnRHR expression level Ligand selectivity Presence of GnRHR splice variants Intrinsic cellular properties [99–101] [73,74,101,103,104] [105–109] [6,110–112] [114,115] [118,119] [61,62] effects of both GnRH-I and GnRH-II [13,67] Thus, one intriguing question is how a common GnRHR may mediate different, even opposite, responses to GnRH in the same cell type ⁄ tissue The reasons are unknown, but several possibilities (as summarized in Table 1) can be envisaged First, the treatment condition may be one determinant of the outcome The pulsatility of GnRH release is necessary for the hormone to stimulate pituitary gonadotropes On the other hand, sustained administration of the peptide brings about a short initial stimulation that is rapidly followed by a decrease in gonadotropin synthesis and secretion [99] In support of this, the signal response is different at different doses It has been shown that pulsatile GnRH stimulates more sustained ERK activity (more than h), whereas continuous infusion of aT3-1 cells with GnRH stimulates short-term (2 h) ERK activity [100] There is also evidence that GnRH treatment stimulates cAMP production at nanomolar concentrations, but has an inhibitory effect at micromolar concentrations [101] It should be pointed out that the nanomolar concentration (0.01–1 nm) corresponds to the physiological circulating level, and the effects caused by this dose range may represent the physiological functions of GnRH [54,102] Second, GnRH action has been shown to be mediated by coupling to different Ga-proteins, depending on the time and dose of exposure [101,103] In general, Gaq and Gas are associated with a stimulatory effect [103], whereas Gai often mediates the antiproliferative and proapoptotic effects of GnRH [73,74] Low GnRH concentrations promote the coupling of GnRHR to Gas [101] High concentrations of GnRH cause a switch in receptor coupling from Gas to Gai [101] Moreover, stimulation of cAMP production by GnRH is through coupling to Gas, whereas inhibition of cAMP production at high concentrations of GnRH is through coupling to Gai [101,104] These findings suggest that the intracellular milieu in different tissues results in differential coupling and different phenotypic effects 5486 Third, multiple GnRH-dependent signaling pathways may occur via different subunits of a single G-protein [105] After ligand-induced dissociation, both the a-subunit and bc-subunits are capable of activating various effectors, such as adenylyl cyclase, phospholipase C, and ion channels, thereby conferring on the receptor the potential for dual signaling [106,107] The effector pathway to be activated is specific to the upstream subunits For instance, whereas the a-subunit of Gi inhibits adenylyl cyclase activity, the bc-subunits may stimulate the activities of some adenylyl cyclase subtypes [108,109] The receptor expression level is also known to be a determinant for different signal outcomes [6,110,111] In gonadotropes, different cell surface densities of GnRHR result in the differential regulation of luteinizing hormone and follicle-stimulating hormone subunit gene expression by GnRH-I [112] We and others have previously shown that low doses of GnRH upregulate the expression of its receptor, whereas high doses decrease it [12,111,113] This difference in regulation suggests that high levels of GnRHR expression may enhance the cellular response to GnRH stimulation, presumably due to more efficient signal amplification or altered signaling through coupling to different G-proteins Moreover, ligand selectivity has been proposed to explain the opposite (stimulatory and inhibitory) effects of GnRH For instance, in positively responding prostate carcinoma cell lines, GnRH-I is more effective than GnRH-II On the other hand, in negatively responding cell lines, GnRH-II is much more effective than GnRH-I Given the short plasma halflife of GnRH, efforts have been made to obtain GnRH analogs, to resist degradation and to increase potency However, the different GnRH agonists may selectively stabilize different receptor-active conformations and therefore different ligand-induced selective signaling pathways [114] In this regard, it has been shown that the highly variable amino acid at position of GnRH plays a discriminating role in selecting the receptor conformational state [115] The presence of splice variants of the GnRHR transcript may be another possible reason for the different or opposite responses to GnRH To date, variant transcripts of GnRHR have been isolated in many species, e.g chicken [116], mouse [117] and human [118] Although these splice variants are totally incapable of ligand binding or signal transduction, they have been implicated in the functional regulation of the wild-type receptor Previous studies have reported their inhibitory activity on full-length GnRHR function [119] This inhibition is specific, augmented by increasing FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS Origin Ovary Prostate Endometrium Endometrium Prostate Breast Breast Ovary Ovary Prostate Cell line CaOV-3 DU-145 HEC-1A Ishikawa LNCaP MCF-7 MDA-MB-435 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS OVCAR-3 SKOV-3 TSU-Pr1 Pleural effusion Ascites Ascites Lymph node Primary carcinoma Primary carcinoma Lymph node Pleural effusion Primary carcinoma Brain Isolation Serous Serous Poorly differentiated Ductal Poorly differentiated Moderately differentiated Well differentiated Well differentiated Ductal Serous Original histology Yes Yes Yes Only achievable with estrogen added Yes Yes Yes Yes Yes Yes Tumorigenicity in nude mice Low High High Low in absence of estrogen High Very low High High Low High Anchorageindependent growth Low High High High Low Very low Low High High Low Invasive capability in vitro Effect of GnRH on motility ⁄ invasion › fl ? ? ? fl – › fl › Effect of GnRH on growth fl fl;– fl;– fl;– fl;– fl fl fl fl ›; – Table Physiological characteristics and responses to GnRH of common cancer cell lines fl, decrease; ›, increase; –, no effect; ?, undetermined [12,13,43,136,137] [13,43,125] [54,55,67,120,127] [68,121,133–135] [56,68,122–124,135] [49,120,127,128] [15,47,48,129–131] [15,30,36,48,129–131] [50,55,67,120,127,128,132] [12,76,125,126] References L W T Cheung and A S T Wong GnRH receptor signaling 5487 GnRH receptor signaling L W T Cheung and A S T Wong amounts of the cotransfected splice variant cDNA and possibly by preventing or diverting the normal processing of GnRHR or enhancing GnRHR degradation [118] Finally, it is also possible that differences in response may be ascribed to the intrinsic properties of the cells The physiological characteristics of the human cancer cell lines mentioned in this minireview are summarized in Table For example, in contrast to SKOV-3 cells, OVCAR-3 cells have low invasive potential Thus, whereas low doses of GnRH-I and GnRH-II can exert a significant invasive effect in OVCAR-3 cells, they fail to stimulate SKOV-3 maximally [12,13] Both GnRH-I and GnRH-II only exert inhibitory effects on SKOV-3 cells at high doses [13] Novel receptor(s) for GnRH in humans? An important issue that remains unresolved in this field is whether one or more other GnRHR subtypes exist in humans The discovery of GnRH-II has stimulated the search for a cognate type II GnRHR Molecular cloning of the type II GnRHR in goldfish, marmoset and monkey has shown that the type II receptor is structurally and functionally distinct from the type I receptor [138–140] In humans, a type II GnRHR has not been found However, a search of the human genome database has revealed a putative type II GnRHR gene on chromosome 1q21.1 [140,141] Expression of this type II GnRHR mRNA has been shown in many human tissues, including endometrium, ovary, placenta, and prostate cancer cells [42,55,140–142] Although these findings suggest that the human type II receptor gene is transcriptionally active, the mRNA is disrupted by a frameshift in coding exon and a premature stop codon in exon 2, suggesting that a conventional seven-transmembrane receptor cannot be translated from this gene The gene also overlaps two flanking genes and displays alternative splicing [143] Thus, the functionality of these human type II GnRHR splice variants and their involvement in transmitting signals from GnRH-II are still in question One noteworthy feature of the primate type II GnRHR is that, unlike the type I receptor, it possesses a C-terminal tail, which is responsible for the receptor’s susceptibility to rapid desensitization and internalization [138,144,145] Finch et al showed that GnRH was able to efficiently inhibit the proliferation of breast cancer cells when engineered with sheep type I GnRHR, but not with Xenopus type II GnRHR [145] This clearly implies that the antiproliferative effect of GnRH is mediated most efficiently by a recep5488 tor that is not rapidly desensitized or internalized There is evidence that GnRH-II may act through the type I GnRHR In monkey pituitary cultures, in which the type II GnRHR is functional, GnRH-II has been found to stimulate gonadotropin secretion exclusively through the type I GnRHR [146] In contrast, other evidence suggests that the neuromodulatory action of GnRH-II on mammalian behavior is not mediated via the type I receptor in musk shrews [147] Thus, it appears that GnRH-II may selectively interact with different GnRHRs to mediate its different actions, presumably due to the structural differences between the two GnRHR subtypes Alternatively, it is possible that the human type II GnRHR may be encoded by a different gene that has yet to be identified Database searches have revealed the presence of more than two other GnRHR genes in the human genome apart from the conventional type I receptor gene [148] These genes are located on separate chromosomes Whether functional, full-length transcripts can be produced from these receptor-like genes remains to be determined Recently, a novel GnRH-II-binding protein, in addition to a conventional GnRHR, has been identified by using photoaffinity labeling with an azidobenzoyl-conjugated GnRH-II in prostate cancer cells [149] Taken together, these observations thus suggest the potential existence of novel receptors for GnRH-I and GnRH-II Concluding remarks This overview shows that GnRH modulates a variety of cellular functions in extrapituitary tissues, such as cell growth, invasion, and angiogenesis However, the effects of GnRH are complex and appear to be cell context dependent The ability of GnRH to elicit very different, even opposite (positive and negative), responses in extrapituitary tissues may arise from differential usage of signal transduction pathways and receptor cross-talk Clearly, further studies are required to unravel this complex signaling network and the coordinated regulatory roles of different factors in specific cellular events during tumorigenesis High-throughput gene profiling and bioinformatics approaches should be helpful to expand this area of research The information may also serve as a basis for investigators in the field to explore the signaling mechanisms of other G-protein-coupled receptors Most studies thus far have only been conducted in cellular models, but in vivo approaches will be essential for a complete understanding of the specific role of each GnRH isoform, including the putative GnRH-III isolated from the human brain [150] Given the clinical FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS L W T Cheung and A S T Wong GnRH receptor signaling utility of GnRH analogs in hormone-dependent diseases, better characterization of GnRH actions and its key molecular effectors is a necessary first step to more effective and perhaps new therapeutic strategies for improving clinical outcome 11 Acknowledgements 12 This work was supported by the Hong Kong Research Grant Council grant 778108 to A S T Wong References Chen A, Yahalom D, Ben-Aroya N, Kaganovsky E, Okon E & Koch Y (1998) A second isoform of gonadotropin-releasing hormone is present in the brain of human and rodents FEBS Lett 435, 199–203 White RB, Eisen JA, Kasten TL & Fernald RD (1998) Second gene for gonadotropin-releasing hormone in humans Proc Natl Acad Sci USA 95, 305–309 Densmore VS & Urbanski HF (2003) Relative effect of gonadotropin-releasing hormone (GnRH)-I and GnRH-II on gonadotropin release J Clin Endocrinol Metab 88, 2126–2134 Kauffman AS (2004) Emerging functions of gonadotropin-releasing hormone II in mammalian physiology and behaviour J Neuroendocrinol 16, 794–806 Schally AV, Comaru-Schally AM & Redding TW (1984) Antitumor effects of analogs of hypothalamic hormones in endocrine-dependent cancers Proc Soc Exp Biol Med 175, 259–281 Peng C, Fan NC, Ligier M, Vaananen J & Leung PC (1994) Expression and regulation of gonadotropinreleasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells Endocrinology 135, 1740–1746 Brus L, Lambalk CB, de Koning J, Helder MN, Janssens RM & Schoemaker J (1997) Specific gonadotrophin-releasing hormone analogue binding predominantly in human luteinized follicular aspirates and not in human pre-ovulatory follicles Hum Reprod 12, 769–773 Choi JH, Gilks CB, Auersperg N & Leung PC (2006) Immunolocalization of gonadotropin-releasing hormone (GnRH)-I, GnRH-II, and type I GnRH receptor during follicular development in the human ovary J Clin Endocrinol Metab 91, 4562–4570 Choi KC, Auersperg N & Leung PC (2001) Expression and antiproliferative effect of a second form of gonadotropin-releasing hormone in normal and neoplastic ovarian surface epithelial cells J Clin Endocrinol Metab 86, 5075–5078 10 Emons G, Pahwa GS, Brack C, Sturm R, Oberheuser F & Knuppen R (1989) Gonadotropin releasing hor- 13 14 15 16 17 18 19 20 21 mone binding sites in human epithelial ovarian carcinomata Eur J Cancer Clin Oncol 25, 215–221 Chien CH, Chen CH, Lee CY, Chang TC, Chen RJ & Chow SN (2004) Detection of gonadotropin-releasing hormone receptor and its mRNA in primary human epithelial ovarian cancers Int J Gynecol Cancer 14, 451–458 Cheung LW, Leung PC & Wong AS (2006) Gonadotropin-releasing hormone promotes ovarian cancer cell invasiveness through c-Jun NH2-terminal kinasemediated activation of matrix metalloproteinase (MMP)-2 and MMP-9 Cancer Res 66, 10902– 10910 Chen CL, Cheung LW, Lau MT, Choi JH, Auersperg N, Wang HS, Wong AS & Leung PC (2007) Differential role of gonadotropin-releasing hormone on human ovarian epithelial cancer cell invasion Endocrine 31, 311–320 Srkalovic G, Wittliff JL & Schally AV (1990) Detection and partial characterization of receptors for [D-Trp6]-luteinizing hormone-releasing hormone and epidermal growth factor in human endometrial carcinoma Cancer Res 50, 1841–1846 Chatzaki E, Bax CM, Eidne KA, Anderson L, Grudzinskas JG & Gallagher CJ (1996) The expression of gonadotropin-releasing hormone and its receptor in endometrial cancer, and its relevance as an autocrine growth factor Cancer Res 56, 2059–2065 Imai A, Ohno T, Iida K, Fuseya T, Furui T & Tamaya T (1994) Presence of gonadotropin-releasing hormone receptor and its messenger ribonucleic acid in endometrial carcinoma and endometrium Gynecol Oncol 55, 144–148 Marinaccio M, Reshkin S, Pinto V & Paradiso A (1994) The estimation of LHRH receptors in the tissue of human leiomyoma, myometrium and endometrium Minerva Ginecol 46, 519–526 Iwashita M, Evans MI & Catt KJ (1986) Characterization of a gonadotropin-releasing hormone receptor site in term placenta and chorionic villi J Clin Endocrinol Metab 62, 127–133 Casan EM, Raga F & Polan ML (1999) GnRH mRNA and protein expression in human preimplantation embryos Mol Hum Reprod 5, 234–239 Boyle TA, Belt-Davis DI & Duello TM (1998) Nucleotide sequence analyses predict that human pituitary and human placental gonadotropin-releasing hormone receptors have identical primary structures Endocrine 9, 281–287 Lin LS, Roberts VJ & Yen SS (1995) Expression of human gonadotropin-releasing hormone receptor gene in the placenta and its functional relationship to human chorionic gonadotropin secretion J Clin Endocrinol Metab 80, 580–585 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5489 GnRH receptor signaling L W T Cheung and A S T Wong 22 Kottler ML, Starzec A, Carre MC, Lagarde JP, Martin A & Counis R (1997) The genes for gonadotropinreleasing hormone and its receptor are expressed in human breast with fibrocystic disease and cancer Int J Cancer 71, 595–599 23 Moriya T, Suzuki T, Pilichowska M, Ariga N, Kimura N, Ouchi N, Nagura H & Sasano H (2001) Immunohistochemical expression of gonadotropin releasing hormone receptor in human breast carcinoma Pathol Int 51, 333–337 24 Eidne KA, Flanagan CA, Harris NS & Millar RP (1987) Gonadotropin-releasing hormone (GnRH)-binding sites in human breast cancer cell lines and inhibitory effects of GnRH antagonists J Clin Endocrinol Metab 64, 425–432 25 Eidne KA, Flanagan CA & Millar RP (1985) Gonadotropin-releasing hormone binding sites in human breast carcinoma Science 229, 989–991 26 Limonta P, Moretti RM, Marelli MM, Dondi D, Parenti M & Motta M (1999) The luteinizing hormonereleasing hormone receptor in human prostate cancer cells: messenger ribonucleic acid expression, molecular size, and signal transduction pathway Endocrinology 140, 5250–5256 27 Tieva A, Stattin P, Wikstrom P, Bergh A & Damber JE (2001) Gonadotropin-releasing hormone receptor expression in the human prostate Prostate 47, 276–284 28 Straub B, Muller M, Krause H, Schrader M, Goessl C, Heicappell R & Miller K (2001) Increased incidence of luteinizing hormone-releasing hormone receptor gene messenger RNA expression in hormone-refractory human prostate cancers Clin Cancer Res 7, 2340–2343 29 Kakar SS, Grizzle WE & Neill JD (1994) The nucleotide sequences of human GnRH receptors in breast and ovarian tumors are identical with that found in pituitary Mol Cell Endocrinol 106, 145–149 30 Emons G, Schroder B, Ortmann O, Westphalen S, Schulz KD & Schally AV (1993) High affinity binding and direct antiproliferative effects of luteinizing hormone-releasing hormone analogs in human endometrial cancer cell lines J Clin Endocrinol Metab 77, 1458–1464 31 Emons G, Ortmann O, Becker M, Irmer G, Springer B, Laun R, Holzel F, Schulz KD & Schally AV (1993) High affinity binding and direct antiproliferative effects of LHRH analogues in human ovarian cancer cell lines Cancer Res 53, 5439–5446 32 Segal-Abramson T, Kitroser H, Levy J, Schally AV & Sharoni Y (1992) Direct effects of luteinizing hormone-releasing hormone agonists and antagonists on MCF-7 mammary cancer cells Proc Natl Acad Sci USA 89, 2336–2339 33 Qayum A, Gullick W, Clayton RC, Sikora K & Waxman J (1990) The effects of gonadotrophin releasing hormone analogues in prostate cancer are mediated 5490 34 35 36 37 38 39 40 41 42 43 44 through specific tumour receptors Br J Cancer 62, 96–99 Pahwa GS, Kullander S, Vollmer G, Oberheuser F, Knuppen R & Emons G (1991) Specific low affinity binding sites for gonadotropin-releasing hormone in human endometrial carcinomata Eur J Obstet Gynecol Reprod Biol 41, 135–142 Thompson MA, Adelson MD & Kaufman LM (1991) Lupron retards proliferation of ovarian epithelial tumor cells cultured in serum-free medium J Clin Endocrinol Metab 72, 1036–1041 Volker P, Grundker C, Schmidt O, Schulz KD & Emons G (2002) Expression of receptors for luteinizing hormone-releasing hormone in human ovarian and endometrial cancers: frequency, autoregulation, and correlation with direct antiproliferative activity of luteinizing hormone-releasing hormone analogues Am J Obstet Gynecol 186, 171–179 Connor JP, Buller RE & Conn PM (1994) Effects of GnRH analogs on six ovarian cancer cell lines in culture Gynecol Oncol 54, 80–86 Slotman BJ, Poels LG & Rao BR (1989) A direct LHRH-agonist action on cancer cells is unlikely to be the cause of response to LHRH-agonist treatment Anticancer Res 9, 77–80 Arencibia JM & Schally AV (2000) Luteinizing hormone-releasing hormone as an autocrine growth factor in ES-2 ovarian cancer cell line Int J Oncol 16, 1009– 1013 Gunthert AR, Grundker C, Bottcher B & Emons G (2004) Luteinizing hormone-releasing hormone (LHRH) inhibits apoptosis induced by cytotoxic agent and UV-light but not apoptosis mediated through CD95 in human ovarian and endometrial cancer cells Anticancer Res 24, 1727–1732 Grundker C, Schlotawa L, Viereck V, Eicke N, Horst A, Kairies B & Emons G (2004) Antiproliferative effects of the GnRH antagonist cetrorelix and of GnRH-II on human endometrial and ovarian cancer cells are not mediated through the GnRH type I receptor Eur J Endocrinol 151, 141–149 Grundker C, Gunthert AR, Millar RP & Emons G (2002) Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation J Clin Endocrinol Metab 87, 1427–1430 Kim KY, Choi KC, Auersperg N & Leung PC (2006) Mechanism of gonadotropin-releasing hormone (GnRH)-I and -II-induced cell growth inhibition in ovarian cancer cells: role of the GnRH-I receptor and protein kinase C pathway Endocr Relat Cancer 13, 211–220 Yano T, Pinski J, Radulovic S & Schally AV (1994) Inhibition of human epithelial ovarian cancer cell FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS L W T Cheung and A S T Wong 45 46 47 48 49 50 51 52 53 54 growth in vitro by agonistic and antagonistic analogues of luteinizing hormone-releasing hormone Proc Natl Acad Sci USA 91, 1701–1705 Yano T, Pinski J, Halmos G, Szepeshazi K, Groot K & Schally AV (1994) Inhibition of growth of OV-1063 human epithelial ovarian cancer xenografts in nude mice by treatment with luteinizing hormone-releasing hormone antagonist SB-75 Proc Natl Acad Sci USA 91, 7090–7094 Schally AV (1999) Luteinizing hormone-releasing hormone analogs: their impact on the control of tumorigenesis Peptides 20, 1247–1262 Imai A, Takagi A, Horibe S, Takagi H & Tamaya T (1998) Fas and Fas ligand system may mediate antiproliferative activity of gonadotropin-releasing hormone receptor in endometrial cancer cells Int J Oncol 13, 97–100 Kleinman D, Douvdevani A, Schally AV, Levy J & Sharoni Y (1994) Direct growth inhibition of human endometrial cancer cells by the gonadotropin-releasing hormone antagonist SB-75: role of apoptosis Am J Obstet Gynecol 170, 96–102 Limonta P, Dondi D, Moretti RM, Maggi R & Motta M (1992) Antiproliferative effects of luteinizing hormone-releasing hormone agonists on the human prostatic cancer cell line LNCaP J Clin Endocrinol Metab 75, 207–212 Dondi D, Limonta P, Moretti RM, Marelli MM, Garattini E & Motta M (1994) Antiproliferative effects of luteinizing hormone-releasing hormone (LHRH) agonists on human androgen-independent prostate cancer cell line DU 145: evidence for an autocrine-inhibitory LHRH loop Cancer Res 54, 4091–4095 Montagnani Marelli M, Moretti RM, Dondi D, Limonta P & Motta M (1997) Effects of LHRH agonists on the growth of human prostatic tumor cells: ‘in vitro’ and ‘in vivo’ studies Arch Ital Urol Androl 69, 257–263 Jungwirth A, Pinski J, Galvan G, Halmos G, Szepeshazi K, Cai RZ, Groot K, Vadillo-Buenfil M & Schally AV (1997) Inhibition of growth of androgenindependent DU-145 prostate cancer in vivo by luteinising hormone-releasing hormone antagonist Cetrorelix and bombesin antagonists RC-3940-II and RC-3950II Eur J Cancer 33, 1141–1148 Kraus S, Levy G, Hanoch T, Naor Z & Seger R (2004) Gonadotropin-releasing hormone induces apoptosis of prostate cancer cells: role of c-Jun NH2terminal kinase, protein kinase B, and extracellular signal-regulated kinase pathways Cancer Res 64, 5736–5744 Enomoto M, Seong JY, Kawashima S & Park MK (2004) Proliferation of TSU-Pr1, a human prostatic carcinoma cell line is stimulated by gonadotropinreleasing hormone Life Sci 74, 3141–3152 GnRH receptor signaling 55 Enomoto M, Endo D, Kawashima S & Park MK (2004) Human type II GnRH receptor mediates effects of GnRH on cell proliferation Zool Sci 21, 763–770 56 Miller WR, Scott WN, Morris R, Fraser HM & Sharpe RM (1985) Growth of human breast cancer cells inhibited by a luteinizing hormone-releasing hormone agonist Nature 313, 231–233 57 Gunthert AR, Grundker C, Olota A, Lasche J, Eicke N & Emons G (2005) Analogs of GnRH-I and GnRH-II inhibit epidermal growth factor-induced signal transduction and resensitize resistant human breast cancer cells to 4OH-tamoxifen Eur J Endocrinol 153, 613–625 58 Sharoni Y, Bosin E, Miinster A, Levy J & Schally AV (1989) Inhibition of growth of human mammary tumor cells by potent antagonists of luteinizing hormone-releasing hormone Proc Natl Acad Sci USA 86, 1648–1651 59 Raga F, Casan EM, Wen Y, Huang HY, BonillaMusoles F & Polan ML (1999) Independent regulation of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-3 in human endometrial stromal cells by gonadotropin-releasing hormone: implications in early human implantation J Clin Endocrinol Metab 84, 636–642 60 Chou CS, Zhu H, Shalev E, MacCalman CD & Leung PC (2002) The effects of gonadotropin-releasing hormone (GnRH) I and GnRH II on the urokinase-type plasminogen activator ⁄ plasminogen activator inhibitor system in human extravillous cytotrophoblasts in vitro J Clin Endocrinol Metab 87, 5594–5603 61 Moretti RM, Montagnani Marelli M, Van Groeninghen JC & Limonta P (2002) Locally expressed LHRH receptors mediate the oncostatic and antimetastatic activity of LHRH agonists on melanoma cells J Clin Endocrinol Metab 87, 3791–3797 62 Moretti RM, Monagnani Marelli M, van Groeninghen JC, Motta M & Limonta P (2003) Inhibitory activity of luteinizing hormone-releasing hormone on tumor growth and progression Endocr Relat Cancer 10, 161– 167 63 Hierowski MT, Wettler O & Schally AV (1984) Plasminogen activator and nuclear androgen receptor in rat prostate tumors after treatment with D-Trp-6-LHRH Biomed Pharmacother 38, 297–303 64 Dondi D, Festuccia C, Piccolella M, Bologna M & Motta M (2006) GnRH agonists and antagonists decrease the metastatic progression of human prostate cancer cell lines by inhibiting the plasminogen activator system Oncol Rep 15, 393–400 65 Gnanapragasam VJ, Darby S, Khan MM, Lock WG, Robson CN & Leung HY (2005) Evidence that prostate gonadotropin-releasing hormone receptors mediate an anti-tumourigenic response to analogue therapy in hormone refractory prostate cancer J Pathol 206, 205–213 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5491 GnRH receptor signaling L W T Cheung and A S T Wong 66 Yates C, Wells A & Turner T (2005) Luteinising hormone-releasing hormone analogue reverses the cell adhesion profile of EGFR overexpressing DU-145 human prostate carcinoma subline Br J Cancer 92, 366–375 67 Enomoto M, Utsumi M & Park MK (2006) Gonadotropin-releasing hormone induces actin cytoskeleton remodeling and affects cell migration in a cell-type-specific manner in TSU-Pr1 and DU145 cells Endocrinology 147, 530–542 68 von Alten J, Fister S, Schulz H, Viereck V, Frosch KH, Emons G & Grundker C (2006) GnRH analogs reduce invasiveness of human breast cancer cells Breast Cancer Res Treat 100, 13–21 69 Chen A, Ganor Y, Rahimipour S, Ben-Aroya N, Koch Y & Levite M (2002) The neuropeptides GnRH-II and GnRH-I are produced by human T cells and trigger laminin receptor gene expression, adhesion, chemotaxis and homing to specific organs Nat Med 8, 1421–1426 70 Parborell F, Irusta G, Rodriguez Celin A & Tesone M (2008) Regulation of ovarian angiogenesis and apoptosis by GnRH-I analogs Mol Reprod Dev 75, 623–631 71 Taylor PD, Hillier SG & Fraser HM (2004) Effects of GnRH antagonist treatment on follicular development and angiogenesis in the primate ovary J Endocrinol 183, 1–17 72 Di Lieto A, De Falco M, Pollio F, Mansueto G, Salvatore G, Somma P, Ciociola F, De Rosa G & Staibano S (2005) Clinical response, vascular change, and angiogenesis in gonadotropin-releasing hormone analogue-treated women with uterine myomas J Soc Gynecol Investig 12, 123–128 73 Kraus S, Naor Z & Seger R (2006) Gonadotropinreleasing hormone in apoptosis of prostate cancer cells Cancer Lett 234, 109–123 74 Imai A, Horibe S, Takagi A & Tamaya T (1997) Gi protein activation of gonadotropin-releasing hormonemediated protein dephosphorylation in human endometrial carcinoma Am J Obstet Gynecol 176, 371–376 75 Kraus S, Benard O, Naor Z & Seger R (2003) c-Src is activated by the epidermal growth factor receptor in a pathway that mediates JNK and ERK activation by gonadotropin-releasing hormone in COS7 cells J Biol Chem 278, 32618–32630 76 Kimura A, Ohmichi M, Kurachi H, Ikegami H, Hayakawa J, Tasaka K, Kanda Y, Nishio Y, Jikihara H, Matsuura N et al (1999) Role of mitogen-activated protein kinase ⁄ extracellular signal-regulated kinase cascade in gonadotropin-releasing hormone-induced growth inhibition of a human ovarian cancer cell line Cancer Res 59, 5133–5142 77 Kim KY, Choi KC, Park SH, Chou CS, Auersperg N & Leung PC (2004) Type II gonadotropin-releasing hormone stimulates p38 mitogen-activated protein 5492 78 79 80 81 82 83 84 85 86 87 88 89 kinase and apoptosis in ovarian cancer cells J Clin Endocrinol Metab 89, 3020–3026 Shah BH, Farshori MP, Jambusaria A & Catt KJ (2003) Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1 ⁄ responses to gonadotropin-releasing hormone receptor activation J Biol Chem 278, 19118–19126 Marshall CJ (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation Cell 80, 179–185 Kao S, Jaiswal RK, Kolch W & Landreth GE (2001) Identification of the mechanisms regulating the differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in PC12 cells J Biol Chem 276, 18169–18177 Pouyssegur J, Volmat V & Lenormand P (2002) Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling Biochem Pharmacol 64, 755–763 Chakraborti S, Mandal M, Das S, Mandal A & Chakraborti T (2003) Regulation of matrix metalloproteinases: an overview Mol Cell Biochem 253, 269–285 Shah BH, Soh JW & Catt KJ (2003) Dependence of gonadotropin-releasing hormone-induced neuronal MAPK signaling on epidermal growth factor receptor transactivation J Biol Chem 278, 2866–2875 Roelle S, Grosse R, Aigner A, Krell HW, Czubayko F & Gudermann T (2003) Matrix metalloproteinases and mediate epidermal growth factor receptor transactivation by gonadotropin-releasing hormone J Biol Chem 278, 47307–47318 Moretti RM, Marelli MM, Dondi D, Poletti A, Martini L, Motta M & Limonta P (1996) Luteinizing hormone-releasing hormone agonists interfere with the stimulatory actions of epidermal growth factor in human prostatic cancer cell lines, LNCaP and DU 145 J Clin Endocrinol Metab 81, 3930–3937 Grundker C, Volker P & Emons G (2001) Antiproliferative signaling of luteinizing hormone-releasing hormone in human endometrial and ovarian cancer cells through G protein alpha(I)-mediated activation of phosphotyrosine phosphatase Endocrinology 142, 2369–2380 Eicke N, Gunthert AR, Emons G & Grundker C (2006) GnRH-II agonist [D-Lys6]GnRH-II inhibits the EGF-induced mitogenic signal transduction in human endometrial and ovarian cancer cells Int J Oncol 29, 1223–1229 Lamharzi N, Halmos G, Jungwirth A & Schally AV (1998) Decrease in the level and mRNA expression of LH-RH and EGF receptors after treatment with LHRH antagonist cetrorelix in DU-145 prostate tumor xenografts in nude mice Int J Oncol 13, 429–435 Montagnani Marelli M, Moretti RM, Mai S, Procacci P & Limonta P (2007) Gonadotropin-releasing hormone agonists reduce the migratory and the invasive FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS L W T Cheung and A S T Wong 90 91 92 93 94 95 96 97 98 behavior of androgen-independent prostate cancer cells by interfering with the activity of IGF-I Int J Oncol 30, 261–271 Marelli MM, Moretti RM, Dondi D, Motta M & Limonta P (1999) Luteinizing hormone-releasing hormone agonists interfere with the mitogenic activity of the insulin-like growth factor system in androgen-independent prostate cancer cells Endocrinology 140, 329–334 Lamharzi N, Schally AV & Koppan M (1998) Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix inhibits growth of DU-145 human androgen-independent prostate carcinoma in nude mice and suppresses the levels and mRNA expression of IGF-II in tumors Regul Pept 77, 185–192 Bifulco G, Miele C, Pellicano M, Trencia A, Ferraioli M, Paturzo F, Tommaselli GA, Beguinot F & Nappi C (2004) Molecular mechanisms involved in GnRH analogue-related apoptosis for uterine leiomyomas Mol Hum Reprod 10, 43–48 Shah BH, Neithardt A, Chu DB, Shah FB & Catt KJ (2006) Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs J Cell Physiol 206, 47–57 Grundker C, Schulz K, Gunthert AR & Emons G (2000) Luteinizing hormone-releasing hormone induces nuclear factor kappaB-activation and inhibits apoptosis in ovarian cancer cells J Clin Endocrinol Metab 85, 3815–3820 Sakamoto Y, Harada T, Horie S, Iba Y, Taniguchi F, Yoshida S, Iwabe T & Terakawa N (2003) Tumor necrosis factor-alpha-induced interleukin-8 (IL-8) expression in endometriotic stromal cells, probably through nuclear factor-kappa B activation: gonadotropin-releasing hormone agonist treatment reduced IL-8 expression J Clin Endocrinol Metab 88, 730–735 Choi JR, Park DW, Choi DS & Min CK (2006) Role of integrin, FAK (focal adhesion kinase) and ERK (extracellular signal regulated kinase) on the suppressed cell proliferation of endometrial cancer cells by GnRH (gonadotropin-releasing hormone) Korean J Fertil Steril 33, 115–123 Chegini N & Kornberg L (2003) Gonadotropin releasing hormone analogue therapy alters signal transduction pathways involving mitogen-activated protein and focal adhesion kinases in leiomyoma J Soc Gynecol Investig 10, 21–26 Maudsley S, Davidson L, Pawson AJ, Freestone SH, Lopez de Maturana R, Thomson AA & Millar RP (2006) Gonadotropin-releasing hormone functionally antagonizes testosterone activation of the human androgen receptor in prostate cells through focal adhesion complexes involving Hic-5 Neuroendocrinology 84, 285–300 GnRH receptor signaling 99 Kaiser UB, Conn PM & Chin WW (1997) Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor-expressing pituitary cell lines Endocr Rev 18, 46–70 100 Haisenleder DJ, Cox ME, Parsons SJ & Marshall JC (1998) Gonadotropin-releasing hormone pulses are required to maintain activation of mitogenactivated protein kinase: role in stimulation of gonadotrope gene expression Endocrinology 139, 3104–3111 101 Krsmanovic LZ, Mores N, Navarro CE, Arora KK & Catt KJ (2003) An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion Proc Natl Acad Sci USA 100, 2969–2974 102 Loumaye E & Catt KJ (1982) Homologous regulation of gonadotropin-releasing hormone receptors in cultured pituitary cells Science 215, 983–985 103 Liu F, Usui I, Evans LG, Austin DA, Mellon PL, Olefsky JM & Webster NJ (2002) Involvement of both G(q ⁄ 11) and G(s) proteins in gonadotropin-releasing hormone receptor-mediated signaling in L beta T2 cells J Biol Chem 277, 32099–32108 104 Krsmanovic LZ, Mores N, Navarro CE, Tomic M & Catt KJ (2001) Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons Mol Endocrinol 15, 429–440 105 Grosse R, Schmid A, Schoneberg T, Herrlich A, Muhn P, Schultz G & Gudermann T (2000) Gonadotropinreleasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q ⁄ 11) proteins J Biol Chem 275, 9193–9200 106 Guo CH, Janovick JA, Kuphal D & Conn PM (1995) Transient transfection of GGH3-1¢ cells [GH3 cells stably transfected with the gonadotropin-releasing hormone (GnRH) receptor complementary deoxyribonucleic acid] with the carboxyl-terminal of beta-adrenergic receptor kinase blocks prolactin release: evidence for a role of the G protein beta gamma-subunit complex in GnRH signal transduction Endocrinology 136, 3031–3036 107 Clapham DE & Neer EJ (1993) New roles for G-protein beta gamma-dimers in transmembrane signalling Nature 365, 403–406 108 Federman AD, Conklin BR, Schrader KA, Reed RR & Bourne HR (1992) Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits Nature 356, 159–161 109 Tang WJ & Gilman AG (1991) Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits Science 254, 1500–1503 110 Kaiser UB, Jakubowiak A, Steinberger A & Chin WW (1993) Regulation of rat pituitary gonadotropin-releasing hormone receptor mRNA levels in vivo and in vitro Endocrinology 133, 931–934 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5493 GnRH receptor signaling L W T Cheung and A S T Wong 111 Kang SK, Cheng KW, Nathwani PS, Choi KC & Leung PC (2000) Autocrine role of gonadotropinreleasing hormone and its receptor in ovarian cancer cell growth Endocrine 13, 297–304 112 Kaiser UB, Sabbagh E, Katzenellenbogen RA, Conn PM & Chin WW (1995) A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone Proc Natl Acad Sci USA 92, 12280–12284 113 McArdle CA, Gorospe WC, Huckle WR & Conn PM (1987) Homologous down-regulation of gonadotropinreleasing hormone receptors and desensitization of gonadotropes: lack of dependence on protein kinase C Mol Endocrinol 1, 420–429 114 Lu ZL, Gallagher R, Sellar R, Coetsee M & Millar RP (2005) Mutations remote from the human gonadotropin-releasing hormone (GnRH) receptor-binding sites specifically increase binding affinity for GnRH II but not GnRH I: evidence for ligand-selective, receptoractive conformations J Biol Chem 280, 29796–29803 115 Lu ZL, Coetsee M, White CD & Millar RP (2007) Structural determinants for ligand–receptor conformational selection in a peptide G protein-coupled receptor J Biol Chem 282, 17921–17929 116 Shimizu M & Bedecarrats GY (2006) Identification of a novel pituitary-specific chicken gonadotropin-releasing hormone receptor and its splice variants Biol Reprod 75, 800–808 117 Zhou W & Sealfon SC (1994) Structure of the mouse gonadotropin-releasing hormone receptor gene: variant transcripts generated by alternative processing DNA Cell Biol 13, 605–614 118 Kottler ML, Bergametti F, Carre MC, Morice S, Decoret E, Lagarde JP, Starzec A & Counis R (1999) Tissue-specific pattern of variant transcripts of the human gonadotropin-releasing hormone receptor gene Eur J Endocrinol 140, 561–569 119 Grosse R, Schoneberg T, Schultz G & Gudermann T (1997) Inhibition of gonadotropin-releasing hormone receptor signaling by expression of a splice variant of the human receptor Mol Endocrinol 11, 1305–1318 120 Bahk JY, Hyun JS, Lee H, Kim MO, Cho GJ, Lee BH & Choi WS (1998) Expression of gonadotropinreleasing hormone (GnRH) and GnRH receptor mRNA in prostate cancer cells and effect of GnRH on the proliferation of prostate cancer cells Urol Res 26, 259–264 121 Chatzistamou I, Schally AV, Varga JL, Groot K, Busto R, Armatis P & Halmos G (2001) Inhibition of growth and metastases of MDA-MB-435 human estrogen-independent breast cancers by an antagonist of growth hormone-releasing hormone Anticancer Drugs 12, 761–768 122 Osborne CK, Hobbs K & Trent JM (1987) Biological differences among MCF-7 human breast cancer cell 5494 123 124 125 126 127 128 129 130 131 132 133 134 lines from different laboratories Breast Cancer Res Treat 9, 111–121 Dickson RB, Bates SE, McManaway ME & Lippman ME (1986) Characterization of estrogen responsive transforming activity in human breast cancer cell lines Cancer Res 46, 1707–1713 Xie D, Miller CW, O’Kelly J, Nakachi K, Sakashita A, Said JW, Gornbein J & Koeffler HP (2001) Breast cancer Cyr61 is overexpressed, estrogen-inducible, and associated with more advanced disease J Biol Chem 276, 14187–14194 Yao JJ, Liu Y, Lacorazza HD, Soslow RA, Scandura JM, Nimer SD & Hedvat CV (2007) Tumor promoting properties of the ETS protein MEF in ovarian cancer Oncogene 26, 4032–4037 Hongo A, Kuramoto H, Nakamura Y, Hasegawa K, Nakamura K, Kodama J & Hiramatsu Y (2003) Antitumor effects of a soluble insulin-like growth factor I receptor in human ovarian cancer cells: advantage of recombinant protein administration in vivo Cancer Res 63, 7834–7839 Webber MM, Bello D & Quader S (1997) Immortalized and tumorigenic adult human prostatic epithelial cell lines: characteristics and applications Part Tumorigenic cell lines Prostate 30, 58–64 Sobel RE & Sadar MD (2005) Cell lines used in prostate cancer research: a compendium of old and new lines – part J Urol 173, 342–359 Kyo S, Nakamura M, Kiyono T, Maida Y, Kanaya T, Tanaka M, Yatabe N & Inoue M (2003) Successful immortalization of endometrial glandular cells with normal structural and functional characteristics Am J Pathol 163, 2259–2269 Tanaka R, Saito T, Ashihara K, Nishimura M, Mizumoto H & Kudo R (2003) Three-dimensional coculture of endometrial cancer cells and fibroblasts in human placenta derived collagen sponges and expression matrix metalloproteinases in these cells Gynecol Oncol 90, 297–304 Sillem M, Prifti S, Koumouridis A & Runnebaum B (1999) Invasiveness corresponds to differentiation rather than to proteinase secretion in endometrial cancer cell lines Eur J Gynaecol Oncol 20, 367–370 Stone KR, Mickey DD, Wunderli H, Mickey GH & Paulson DF (1978) Isolation of a human prostate carcinoma cell line (DU 145) Int J Cancer 21, 274– 281 Honjo Y, Nangia-Makker P, Inohara H & Raz A (2001) Down-regulation of galectin-3 suppresses tumorigenicity of human breast carcinoma cells Clin Cancer Res 7, 661–668 Mukhopadhyay R, Theriault RL & Price JE (1999) Increased levels of alpha6 integrins are associated with the metastatic phenotype of human breast cancer cells Clin Exp Metastasis 17, 325–332 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS L W T Cheung and A S T Wong 135 Thompson EW, Paik S, Brunner N, Sommers CL, Zugmaier G, Clarke R, Shima TB, Torri J, Donahue S, Lippman ME et al (1992) Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines J Cell Physiol 150, 534–544 136 Hamilton TC, Young RC, McKoy WM, Grotzinger KR, Green JA, Chu EW, Whang-Peng J, Rogan AM, Green WR & Ozols RF (1983) Characterization of a human ovarian carcinoma cell line (NIH:OVCAR-3) with androgen and estrogen receptors Cancer Res 43, 5379–5389 137 Maeda T, Tashiro H, Katabuchi H, Begum M, Ohtake H, Kiyono T & Okamura H (2005) Establishment of an immortalised human ovarian surface epithelial cell line without chromosomal instability Br J Cancer 93, 116–123 138 Millar R, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, Ott T, Millar M, Lincoln G, Sellar R et al (2001) A novel mammalian receptor for the evolutionarily conserved type II GnRH Proc Natl Acad Sci USA 98, 9636–9641 139 Illing N, Troskie BE, Nahorniak CS, Hapgood JP, Peter RE & Millar RP (1999) Two gonadotropinreleasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus) Proc Natl Acad Sci USA 96, 2526–2531 140 Neill JD, Duck LW, Sellers JC & Musgrove LC (2001) A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates Biochem Biophys Res Commun 282, 1012–1018 141 Neill JD (2002) GnRH and GnRH receptor genes in the human genome Endocrinology 143, 737–743 142 Eicke N, Gunthert AR, Viereck V, Siebold D, Behe M, Becker T, Emons G & Grundker C (2005) GnRHII receptor-like antigenicity in human placenta and in cancers of the human reproductive organs Eur J Endocrinol 153, 605–612 GnRH receptor signaling 143 Morgan K, Conklin D, Pawson AJ, Sellar R, Ott TR & Millar RP (2003) A transcriptionally active human type II gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the antisense orientation on chromosome 1q.12 Endocrinology 144, 423–436 144 Pawson AJ, Faccenda E, Maudsley S, Lu ZL, Naor Z & Millar RP (2007) Mammalian type I GnRH receptors undergo slow, constitutive, agonist-independent internalization Endocrinology 149, 1415–1422 145 Finch AR, Green L, Hislop JN, Kelly E & McArdle CA (2004) Signaling and antiproliferative effects of type I and II gonadotropin-releasing hormone receptors in breast cancer cells J Clin Endocrinol Metab 89, 1823–1832 146 Okada Y, Murota-Kawano A, Kakar SS & Winters SJ (2003) Evidence that gonadotropin-releasing hormone (GnRH) II stimulates luteinizing hormone and folliclestimulating hormone secretion from monkey pituitary cultures by activating the GnRH I receptor Biol Reprod 69, 1356–1361 147 Kauffman AS, Wills A, Millar RP & Rissman EF (2005) Evidence that the type-2 gonadotrophin-releasing hormone (GnRH) receptor mediates the behavioural effects of GnRH-II on feeding and reproduction in musk shrews J Neuroendocrinol 17, 489–497 148 van Biljon W, Wykes S, Scherer S, Krawetz SA & Hapgood J (2002) Type II gonadotropin-releasing hormone receptor transcripts in human sperm Biol Reprod 67, 1741–1749 149 Maiti K, Oh DY, Moon JS, Acharjee S, Li JH, Bai DG, Park HS, Lee K, Lee YC, Jung NC et al (2005) Differential effects of gonadotropin-releasing hormone (GnRH)-I and GnRH-II on prostate cancer cell signaling and death J Clin Endocrinol Metab 90, 4287–4298 150 Yahalom D, Chen A, Ben-Aroya N, Rahimipour S, Kaganovsky E, Okon E, Fridkin M & Koch Y (1999) The gonadotropin-releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform FEBS Lett 463, 289–294 FEBS Journal 275 (2008) 5479–5495 ª 2008 The Authors Journal compilation ª 2008 FEBS 5495 ... representation of GnRHR signaling in extrapituitary tissues Binding of GnRH to GnRHR triggers several intracellular signaling cascades and cross-talk with mitogenic signaling, depending on the cell... protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K), and nuclear factor kappa B (NF-jB) signaling The GnRH- induced signaling pathways in extrapituitary tissues are shown schematically in. .. 84, 285–300 GnRH receptor signaling 99 Kaiser UB, Conn PM & Chin WW (1997) Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor- expressing pituitary cell lines Endocr Rev