Gonadotropin-Releasing Hormone Receptor Levels and Cell Context Affect Tumor Cell Responses to Agonist in Vitro and in Vivo

Research Article

Gonadotropin-Releasing Hormone Receptor Levels and Cell Context Affect Tumor Cell Responses to Agonist In vitro and In vivo

Kevin Morgan,1 Alan J. Stewart,1 Nicola Miller,1 Peter Mullen,2 Morwenna Muir,2 Michael Dodds,2 Federico Medda,1 David Harrison,2 Simon Langdon,2 and Robert P. Millar1

1Medical Research Council Human Reproductive Sciences Unit and 2Edinburgh Breakthrough Research Unit, Edinburgh Cancer Research Centre, Edinburgh, United Kingdom

Abstract uterine cancer (4), and premenopausal breast cancer (5, 6) has been studied in small (3, 4) or moderately sized clinical trials (2) since Activation of gonadotropin-releasing hormone (GnRH) receptors inhibits proliferation of transformed cells derived from the late 1980s. Larger data sets indicate that a relatively poor, reproductive tissues and in transfected cell lines. Hence, GnRH widely variable, yet favorable additive effect on response rates can receptors represent a therapeutic target for direct action of occur when GnRH is used in combination therapy (5, 6). Most GnRH analogues on certain proliferating cells. However, more studies have focused on patients with advanced cancer, with fewer cell biological data are required to develop this particular analyzing the effects of early-stage treatment. A recent meta- application of GnRH analogues. Therefore, we compared the analysis encompassing a large sample size (11,900 cases) reported effects of GnRH receptor activation in transfected HEK293 small benefits (12% additional decrease in disease recurrence rate) associated with GnRH analogues in the treatment of steroid cells (HEK293[SCL60]) with transfected human ovarian cancer cell lines SKOV3 and EFO21, human hepatoblastoma receptor–positive breast cancer when used in combination with tamoxifen (5). On the other hand, GnRH agonists are widely and HepG2 cells, and rat neuroblastoma B35 cells. Marked differences in receptor levels, magnitude of inositol phosphate effectively used clinically to suppress androgen levels and extend generation, and dynamics of inositol phosphate turnover survival in men with advanced prostate cancer (7). occurred in the different cells. Activation of GnRH receptors, In certain tumors, a direct antiproliferative effect of GnRH expressed at high or moderate levels, inhibited the growth of receptor activation on malignant cells represents an added, but currently unexploited, benefit of GnRH analogue therapy (8–16). HEK293[SCL60] and B35 cells, respectively. Western blotting detected markers of apoptosis [cleaved poly(ADP-ribose) However, the extent to which this direct action may be applicable to cancer therapy remains uncertain. The incidence of GnRH polymerase, caspase-9] in HEK293[SCL60] and B35 following 6 receptor–positive tumors is undefined and many phenotypic treatment with 100 nmol/L D-Trp -GnRH-I. Cell growth inhibition was partially or completely rescued with inhibitor variables probably influence the effect of GnRH receptor activation Q-VD-OPh or Ro32-0432. Low levels of GnRH receptor on cell fate, including oncogenic mechanisms likely to mediate expression in transfected SKOV3, EFO21, or HepG2 activated resistance to GnRH action. intracellular signaling but did not induce apoptosis or signi- One concern is that cell types in the mammalian body express GnRH receptors in different context. For instance, high levels of ficantly affect cell proliferation. Tumor xenografts prepared 6 receptor occur in pituitary gonadotropes and in certain neuronal from HEK293 regressed during treatment with D-Trp - [SCL60] cells within the central nervous system whereas much lower levels GnRH-I and growth of xenografts derived from transfected of receptor expression occur in peripheral reproductive tissues (17), B35 was slowed. SKOV3 xenografts were not growth inhibited. including pathological specimens (8, 18). More studies are required Therefore, differences in levels of GnRH receptor and signaling to address the practical significance of variations in receptor differentially affect the apoptotic machinery within cell lines expression levels and other cell-specific differences to enable and contribute to the cell type–specific effects of GnRH on improved application of GnRH analogues in the treatment of growth. Further studies should exploit the growth-inhibitory proliferative disease. potential of GnRH receptor activation in abnormal cells in Thus far, diverse in vitro studies using cells that endogenously diseased human tissues. [Cancer Res 2008;68(15):6331–40] express low levels of GnRH receptor have confused the evaluation of the applicability of GnRH-mediated cell growth inhibition. For Introduction instance, low levels of receptor expression have been described in a Gonadotropin-releasing hormone (GnRH) analogue therapy is number of cell lines derived from human reproductive tissue potentially useful in the treatment of certain malignancies, malignancies [including breast (MCF-7 and MDA-MB-231), ovary particularly those that are sex steroid hormone dependent, due (OV-1063, OVCAR-3, SKOV3, EFO21, EFO27, and Caov-3), uterine to the sex steroid–lowering effects of GnRH administration. epithelium (Hec1A, Ishikawa, and RL95-2), and prostate (AT-1, 6 Application of long-acting analogues such as D-Trp -GnRH-I DU145, and LNCaP); refs. 8–16]. Very significant cell growth– superagonist (1, 2) in second-line therapy for ovarian cancer (3), inhibitory effects of GnRH analogues on all these cell lines have been reported (often >30% inhibition within 5–9 days). Effects on transformed cells from nonreproductive tissues, including liver,

Requests for reprints: Kevin Morgan, Medical Research Council Human pancreas, and melanoma, have also been described (19, 20). Growth Reproductive Sciences Unit, The Queen’s Medical Research Institute, 47 Little France of some of these cells as tumor xenografts in nude mice can be Crescent, Edinburgh EH16 4TJ, United Kingdom. Phone: 44-131-242-6247; Fax: 44-131- inhibited with GnRH analogues. 242-6197; E-mail: [email protected]. I2008 American Association for Cancer Research. However, contradictory data exist in that the absence of doi:10.1158/0008-5472.CAN-08-0197 response to GnRH analogues has been described for some cell www.aacrjournals.org 6331 Cancer Res 2008; 68: (15). August 1, 2008

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Cancer Research lines in vitro (10) or in tumor xenograft studies (21). One possibility Collection. Nontransfected EFO21 cells (39) were a gift from Prof. Gu¨nter is that fluctuations in levels of receptor expression occur during Emons (Georg-August-University, Go¨ttingen, Germany). HEK293 cells cell passage in vitro or as a consequence of differences in culture expressing the rat GnRH receptor HEK293[SCL60] cells were generated by conditions. Certain cells (e.g., LNCaP) show either a proliferative or our colleagues in our laboratory (40). Isolation of cell clones stably expressing rat GnRH receptor. Stably an antiproliferative response to GnRH analogue depending on transfected cell lines were prepared following electroporation or Fugene 6 analogue concentration (15). Hormones such as estrogen (22), transfection (Roche) of rat GnRH receptor cDNA in pcDNA3.1 (Invitrogen) insulin-like growth factors, or members of the epidermal growth into cells followed by G418 antibiotic selection and clone isolation. 125 5 6 factor family may counteract the growth-inhibitory effects of GnRH binding assay. I-radiolabeled (His -D-Tyr )-GnRH-I analogue GnRH (17). bound to cells in 12-well plates was displaced with different concentrations The relevance of comparisons between effects of GnRH on of unlabeled native GnRH-I, each in triplicate. different cell lines in vitro or in xenograft experiments to the The number of cells per well was determined using a hemocytometer clinical situation is currently difficult to interpret. Nevertheless, following trypsinization of wells from a plate prepared in parallel. Binding in vitro studies have implicated various intracellular signaling data were analyzed using Prism software (GraphPad). pathways in GnRH analogue–mediated cell growth inhibition Inositol phosphate assay. Cells in 12-well plates were assayed for generation of 3H-radiolabeled inositol phosphate following treatment with (23–31), with cell cycle arrest and apoptosis clearly established as GnRH or phospholipase C activator m-3m3FBS at various concentrations outcomes in target cells. However, whether different cell types (each in triplicate) using a method adapted for batch chromatography with share a common mechanism for GnRH-mediated growth inhibition AG 1-X8 resin (Bio-Rad) in 12 Â 75 mm plastic tubes. The number of cells is not clear. One way to rigorously investigate this question, and per well was determined as described above. other uncertainties, is to examine different cell types engineered in In vitro growth assay. Cells growing in 12-well plates (200,000 per well parallel to express the GnRH receptor by genetic manipulation. In with 2-mL medium) were treated with GnRH in triplicate and growth was transfected cells, activation of the mammalian type I GnRH monitored using the sulforhodamine B staining assay (41). Growth rates for receptor, including the human isoform (32), reproducibly inhibits untreated cells in 12-well plates were also measured by daily sampling in growth in vitro, largely irrespective of passage number under duplicate over a period of 120 h by manual counting with a hemocytometer standard culture conditions and without the need to manipulate following trypsinization. The data were analyzed using Prism software. Western blotting. Cells were cultured in six-well plates for short time- steroid hormone content or other culture components. However, course experiments (i.e., for treatments up to 6–8 h) or in 6-cm dishes for recently studied cell lines, either transfected with GnRH receptor time courses up to 5 d. Cells were lysed in ice-cold NP40 lysis buffer. Cellular cDNA (e.g., HEK293 and MCF-7) or isolated from transgenic mice fractions were collected following 10-min centrifugation at 14,000 rpm and (e.g., LhT2 or aT3-1 embryonic gonadotropes), express the GnRH mixed with SDS-PAGE loading buffer. For quantitative analysis, treatments receptor at relatively high levels, unlike the situation in cells were done in triplicate. Western blotting was done with SDS-PAGE mini- derived from tumors. Therefore, levels of GnRH receptor expression gels (Invitrogen), prestained broad-range molecular weight markers (Bio- at the cell surface have been carefully titrated in MCF-7 and PC3 Rad), and polyvinylidene difluoride membrane (Perkin-Elmer). Rabbit cells using an adenoviral vector. Cell-surface receptor level was polyclonal antibodies were purchased from Cell Signaling Technology: shown to be a major influence on the extent of GnRH-mediated anti–phospho-extracellular signal–regulated kinase (ERK)-1/2, anti–total growth inhibition in vitro (33, 34). ERK1/2, anti–phospho-p38, anti–cleaved poly(ADP-ribose) polymerase (PARP; human or rat specific), anti–cleaved caspase-9, and anti–phospho- The behavior of cell lines stably transfected with GnRH receptor h in vitro Fas-associated death domain (FADD); -actin was from Abcam. Specific has not previously been studied simultaneously and antibody binding was detected by enhanced chemifluorescence with in vivo , where pharmacokinetic and systemic factors may influence alkaline phosphatase–conjugated secondary antibody (Sigma) and en- cell proliferation. Therefore, we compared the effects of GnRH hanced chemifluorescence substrate (GE Healthcare) on a Typhoon receptor activation on the growth of five different transfected phosphoimager (Amersham Biosciences). Bands were measured using cell lines stably expressing rat GnRH receptor over a range of ImageQuant software (Amersham Biosciences). levels. The cells were studied both in vitro and in vivo with the Tumor xenograft experiments. Cloned cell lines were expanded in vitro aim of identifying factors important in determining the effects of in the presence of 500 Ag/mL G418 before implantation into nude mice. Cells 6 D-Trp -GnRH-I on growth. We confirmed that the level of GnRH (5–10 million) were implanted s.c. (bilaterally) into the flanks of groups of receptor expression correlates with the extent of growth inhibition adult female nude mice. At least five animals per group were studied. Pharmacologic treatment was initiated when tumors were 50 to 100 mm3 in both in vitro and in vivo, and we established that in vitro growth size. Tumor volumes were measured in two diameters using calipers and inhibition can be prevented in a cell type–specific fashion using volumes were calculated (V = p Â D Â d2 /6,whereD and d represent the exogenous chemical inhibitors of signaling. The results suggest larger and smaller diameters, respectively). Mean tumor volumes were strategies to develop more precisely targeted applications of GnRH measured and expressed as a ratio of the value on day 0 of treatment. analogues on appropriate GnRH receptor–positive cells. Statistical analyses. Statistical analysis of data was done using online software (Simple Interactive Statistical Analysis3 and the SD calculator4). Materials and Methods 6 Results Reagents. Most chemicals were purchased from Sigma: D-Trp -GnRH-I, 6 sulforhodamine B, and 8-bromo-cyclic AMP (8-Br-cAMP). D-Trp -GnRH-I Isolation of cell lines expressing rat GnRH receptor. B35, was dissolved in 20% propylene glycol (Sigma). SKOV3, EFO21, and HepG2 cells were transfected with a pcDNA3.1 Certain reagents were from Calbiochem: Ro32-0432, Q-VD-OPh, PD98059, expression construct containing rat GnRH receptor cDNA. SB203580, rottlerin, fumonisin B1, 2-aminoethoxydiphenylborate (2-APB), Following selection with G418, individual colonies were picked and m-3m3FBS. Radioactive reagents, Na125I and myo[3H]inositol, were from Amersham. Cell culture. Cells were grown in DMEM containing 10% FCS, glutamine, and antibiotics. Nontransfected cell lines HEK293 (35), B35 (36), SKOV3 (37), 3 http://home.clara.net/sisa/ and HepG2 (38) were obtained from the American Type Tissue Culture 4 http://www.csgnetwork.com/stdeviationcalc.html

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. GnRH Receptor Levels and Effects on Tumor Growth and expanded. Cell clones were screened for GnRH receptor 22.7 hours; B35-20, 20.7 hours; B35-2, 20.9 hours; and SKOV3-4, 125 6 expression using a I-radioligand binding assay and compared 25.6 hours. Following treatment with D-Trp -GnRH-I or other with HEK293[SCL60] and aT3-1 cells. Stability of receptor expression chemical agents at zero time point, cells were fixed in situ at was verified over the course of subsequent cell passages. Trans- relevant time points, stained with sulforhodamine B, dissolved in fected cell clones exhibited differences in relative levels of receptor 10 mmol/L Tris (pH 10.5), and quantified by 96-well plate expression (Fig. 1). Nontransfected cell lines did not possess spectrophotometry (absorbance at 540 nm). Analysis of cell detectable endogenous specific binding (displaceable radioligand treatments done in triplicate enabled detection of statistically binding never exceeded 1% that observed in transfected cells, see significant differences in cell growth relative to vehicle-treated cells dashed line in Fig. 1). HEK293[SCL60] cells expressed f8.6-fold more (P < 0.02), provided the differences were z10%. Smaller differences GnRH receptor at the cell surface than transfected SKOV3-clone in growth were not usually statistically significant. 4 cells (SKOV3-4), which expressed the lowest level of receptor in GnRH elicited significant growth inhibition for HEK293[SCL60] and the range of cell clones studied here. Rat neuronal cell clones B35- transfected B35 cells, but not for transfected EFO21, SKOV3, or 2b, B35-20, and B35-2 expressed 5.1-, 2.3-, and 1.9-fold higher levels HepG2 cells (Fig. 2). Typically, HEK293[SCL60] cell growth was of receptor per cell than SKOV3-4. Clones EFO21-1 and HepG2-3 inhibited by 70% relative to vehicle-treated cells within 96 hours expressed only slightly more receptor per cell than SKOV3-4 (30% (i.e., sulforhodamine B staining was 30% that of controls). Growth of 125 5 6 and 10%, respectively). Displacement of I-His D-Tyr -GnRH-I B35-2b cells was inhibited by 85% relative to vehicle-treated cells with unlabeled GnRH-I exhibited a sigmoidal curve consistent with after 96 hours. B35-20 and B35-2 cells were growth inhibited by 22% the presence of a single high-affinity receptor in all transfected cell to 23% after 96 hours. Dose-response analysis indicated that 6 clones studied. The ratio [total specific binding/nonspecific 5 nmol/L D-Trp -GnRH-I was sufficient to induce growth inhibition 6 binding] per cell was high in each case: HEK293[SCL60] ratio, 50; as effectively as 25 or 100 nmol/L D-Trp -GnRH-I. For HEK293[SCL60] B35-2b, 18; B35-20, 29; B35-2, 14; EFO21-1, 30; SKOV3-4, 21; and and B35-2b, relative growth inhibition was detectable within HepG2-3, 9. 24 hours and was cumulative during the 96-hour time course. 6 Effects of GnRH on cell growth in vitro. Cell growth was A brief exposure to D-Trp -GnRH-I (10 min) was sufficient to elicit measured following culture in 12-well plates for periods of time up growth inhibition but continuous exposure was marginally more to 96 or 120 hours. Each cell type exhibited exponential growth for effective. Vehicle-treated cell cultures became confluent within 72 hours followed by a plateau in growth rate. Optimal doubling 96 hours but cell viability was not compromised following further times for growth in 12-well plates, measured during the incubation for at least 24 hours without the need to change culture exponential phase, were HEK293[SCL60], 21.4 hours; B35-2b, medium. GnRH-stimulated production of inositol phosphate. Clones exhibiting specific GnRH binding were characterized by [3H]inositol phosphate assay. The amount of inositol phosphate production reflected the level of receptor expression determined by binding assay (Fig. 3). HEK293[SCL60] cells generated 3- to 8-fold more inositol phosphate per cell than transfected B35 cells and 11- to 12-fold more inositol phosphate than transfected SKOV3-4 or EFO21-1 cells. The HepG2-3 clone exhibited weakest inositol phosphate production. Maximum relative levels of inositol phosphate production per cell after 90 minutes of stimulation were HEK293[SCL60], 11.4; B35-2b, 3.8; B35-2, 2.4; B35-20, 1.6; EFO21-1, 1.0; SKOV3-4, 0.6; and HepG2-3, 0.3. The maximum levels of [3H]inositol phosphate were at least 2-fold above basal in cells expressing low levels of GnRH receptor (i.e., EFO21-1, SKOV3-4, and HepG2-3) and were much higher than basal in HEK293[SCL60] cells (at least 50- to 70-fold) and B35 clones (3- to 6-fold). The dynamics for inositol phosphate generation were similar between cell clones, reaching a peak at f90 minutes after receptor activation. On replacement of culture medium to remove GnRH agonist and to release inositol phosphatase inhibition (GnRH and LiCl washed out with DMEM), inositol phosphate levels returned to basal in B35-20 cells within 2 to 2.5 hours, but did not return to Figure 1. GnRH binding to transfected cell lines stably expressing rat GnRH receptor compared with receptor binding in mouse aT3-1 gonadotrope cells. basal levels in HEK293[SCL60], B35-2b, or B35-2 cells. In these clones, Relative levels of cell-surface receptor per cell were determined in triplicate by inositol phosphate remained 9.9-, 2.0-, and 1.5-fold above basal, 125 I-GnRH binding assay. Nonspecific binding was subtracted from maximum respectively, for at least another hour (i.e., 3–3.5 hours in total after binding before calculation relative to SKOV3-4. The highest relative level occurred in HEK293 clone SCL60 (HEK293[SCL60] ). Three clones derived removal of GnRH and LiCl). 6 from transfected rat neuroblastoma B35cell line expressed different levels of D-Trp -GnRH-I was more potent than native GnRH-I in receptor. Transfected ovarian cancer cell lines EFO21-1 and SKOV3-4 and a transfected HepG2 clone expressed only low levels of GnRH receptor. stimulation of inositol phosphate production. In HEK293[SCL60] Representative data depicting specific binding compiled from binding assays cells, 5 nmol/L native GnRH-I elicited 18% of maximal inositol 6 done in triplicate. Maximum specific binding per cell was 9-fold above nonspecific phosphate production whereas 5 nmol/L D-Trp -GnRH-I elicited binding in HepG2-3 cells, compared with 50-fold above nonspecific binding in 90% of maximum inositol phosphate production. In B35-2 cells, HEK293[SCL60] cells. Very low levels of displaceable radioligand binding were detected in nontransfected cells (dashed line). 5 nmol/L native GnRH-I elicited 47% of maximal inositol www.aacrjournals.org 6333 Cancer Res 2008; 68: (15). August 1, 2008

6 Figure 2. Effects of 100 nmol/L D-Trp -GnRH-I on in vitro growth of cells expressing rat GnRH receptor. Cells in 12-well plates were treated on day 0 with either 6 vehicle or GnRH analogue (100 nmol/L D-Trp -GnRH-I, triptorelin). Plates were fixed, stained with sulforhodamine B, and quantified spectrophotometrically following cell growth for up to 4 d. Growth of HEK293[SCL60] cells and B35clones was significantly inhibited, but SKOV3-4, EFO21-1, and HepG2-3 were not growth inhibited. Representative data of assays done in triplicate.

6 phosphate production, whereas 5 nmol/L D-Trp -GnRH-I elicited Manipulation of GnRH effects in vitro. In some experiments, 89% of maximum inositol phosphate production (data not shown). cells were treated with a combination of a signal-modulatory 6 A phospholipase C activator, m-3m3FBS, elicited a small additive compound plus D-Trp -GnRH-I (Table 1). Inhibition of protein effect on production of inositol phosphate in HEK293[SCL60] cells kinase C (PKC) using Ro32-0432 rescued transfected HEK293[SCL60] 6 treated with D-Trp -GnRH-I over the first 5 to 30 minutes of cells from GnRH-mediated growth inhibition (Table 1; Fig. 5). stimulation (30% elevation), followed by marked inhibition of Dose-response analysis indicated that 100 nmol/L Ro32-0432 further inositol phosphate production (62% less inositol phosphate enabled complete protection (100% rescue) and attenuated the 6 relative to cells treated with D-Trp -GnRH-I alone for 90 minutes). appearance of cleaved PARP (Fig. 5B). The general caspase 6 An additive effect of m-3m3FBS plus D-Trp -GnRH-I on production inhibitor Q-VD-OPh (20 Amol/L) enabled partial rescue of these 6 of inositol phosphate was also detectable in SKOV3-4 cells (2.7-fold cells in the presence of 100 nmol/L D-Trp -GnRH-I (40% more cell relative increase in inositol phosphate at 90-minute stimulation) growth after 96 hours compared with cells treated with GnRH but there was no feedback inhibition of phospholipase C in these agonist alone). However, transfected B35 cell clones were partially 6 6 cells relative to cells treated with D-Trp -GnRH-I alone (data not protected from the effects of D-Trp -GnRH-I by Q-VD-OPh (also shown). 40% more cell growth for each clone at 96 hours) but were not GnRH receptor–activated protein kinase signaling in protected by Ro32-0432. different cell lines. Treatment with GnRH elicited rapid transient Certain agents exerted an additive growth-inhibitory effect when 6 phosphorylation of ERK1/2 in transfected HEK293[SCL60], EFO21-1, combined with D-Trp -GnRH-I whereas others had no effect (see SKOV3-4, and HepG2-3 cells. In contrast, transfected B35 cells Table 1). Growth of SKOV3-4 cells was inhibited by treatment with exhibited transient dephosphorylation of ERK1/2 (Fig. 4). Rapid the phospholipase C activator m-3m3FBS (60 Amol/L), and initial transient activation of the stress-activated protein kinase cotreatment with Trp6-GnRH-I elicited a small poorly reproducible p38 was readily detectable in HEK293[SCL60] cells but not in the additive effect (up to 10% increase in growth inhibition after other transfected cell lines. Elevated levels of phosphorylated p38 96 hours; data not shown). The mitogen-activated protein (MAP) were detectable in both HEK293[SCL60] and B35-2b cells at later kinase inhibitors PD98059 and SB203580 did not rescue cells 6 6 stages of exposure to D-Trp -GnRH-I (48–96 hours; Fig. 4C). from the growth-inhibitory effects of 100 nmol/L D-Trp -GnRH-I Time course for detection of markers of apoptosis following (Table 1; Fig. 5C and D). GnRH treatment. Elevated levels of cleaved PARP were readily Effects of GnRH on tumor growth in vivo. Cells were detectable in HEK293[SCL60] cells following 24 to 48 hours of implanted s.c. into the flanks of female athymic nude mice and 6 treatment with D-Trp -GnRH-I. Small elevations in the levels of tumors were derived. Tumors were further propagated by cleaved caspase-9 and phosphorylated FADD were also detectable xenografting. Growth of transfected HEK293[SCL60] xenografts could 6 in these cells (Fig. 4C). Much lower levels of cleaved PARP were be inhibited with daily i.p. doses of 10-Ag D-Trp -GnRH-I, resulting detectable in B35-2b cells compared with HEK293[SCL60]. Elevations in markedly reduced tumor growth rate within 7 days and 6 in these markers attributable to D-Trp -GnRH-I treatment were not persisting for at least 25 days (Fig. 6). Growth of xenografts detected in EFO21-1 or SKOV3-4 cells. However, elevation in the prepared from untransfected HEK293 cells was not affected by 6 level of cleaved PARP was detected in SKOV3-4 cells following D-Trp -GnRH-I (data not shown). A much less dramatic but treatment with m-3m3FBS (data not shown). significant inhibition of tumor growth was observed with B35-2b

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. GnRH Receptor Levels and Effects on Tumor Growth and B35-2 cell xenografts. Transfected B35-20 and SKOV3-4 cell account for loss of low-level endogenous GnRH receptor expression xenografts did not exhibit significant growth inhibition. Attempts in these cell lines. Hence, stably transfected cells in which the level to generate tumors using transfected EFO21-1 cells failed. of receptor expression can be verified represent a good tool for functional studies. The rat GnRH receptor is particularly amenable to such studies because cell clones possessing different levels of Discussion surface receptor can be readily isolated and the receptor possesses The aim of this study was to engineer new sublines of cells pharmacologic properties similar to the human receptor. expressing different levels of rat GnRH receptor at the cell surface and In our cell clones, levels of rat GnRH receptor expression and to use them to assess factors influencing GnRH-mediated cell receptor-stimulated inositol phosphate production both correlated growth inhibition in vitro and in vivo. Our data confirm and extend with the extent of cell growth inhibition in vitro (Figs. 2 and 3). previous in vitro data obtained with transfected HEK293 cells, MCF-7, Relative growth inhibition ranged from 12% to 80% following 6 and PC3 cells (26, 28, 32–34) and contribute some novel observations. 96 hours of treatment with triptorelin (D-Trp -GnRH-I), depending Three independent clones of rat B35 neuroblastoma cells (B35- on the particular clone, and these differences were reflected by the 2b, B35-2, and B35-20) and a derivative of the human ovarian magnitude of inositol phosphate production elicited by GnRH in cancer cell line SKOV3 (SKOV3-4) were informative in in vitro and each clone. in vivo studies done in parallel with HEK293[SCL60] cells (26, 28). GnRH receptor–activated signaling varied between cell types. a HEK293[SCL60] cells exhibited higher GnRH binding than T3-1 B35 cells exhibited a different pattern of GnRH-stimulated ERK gonadotrope cells (Fig. 1). In contrast, the B35 and SKOV3 cell clones phosphorylation compared with the other cell types (a variable and possessed moderate or low levels of cell-surface GnRH receptor transient dephosphorylation rather than clear-cut elevated phos- (Fig. 1). The functional effects of setting different levels of GnRH phorylation; Fig. 4). However, the role of ERK phosphorylation receptor expression using recombinant DNA have hitherto been in determining the cellular growth response remains uncertain described only in in vitro studies using MCF-7 and PC3 cells (33, 34). (30, 43). Nevertheless, cell growth inhibition characteristically Contrary to previous reports, we could not verify endogenous involved elevation in levels of cleaved PARP in both HEK293[SCL60] GnRH receptor expression in nontransfected cell lines (EFO21, and B35 cells (Fig. 4). SKOV3, and HepG2) using binding assay (Fig. 1), inositol phosphate Elevated levels of cleaved caspase-3 and cleaved PARP have production, MAP kinase activation, or in vitro growth inhibition previously been described in HEK293[SCL60] cells following treat- assays. Differences in passage number, culture conditions, or ment with GnRH (28). The levels of cleaved PARP in HEK293[SCL60] trafficking (42) of the human GnRH receptor to the cell surface may and B35-2b cells reflected the level of GnRH receptor expression and

Figure 3. A, time courses for accumulation of 3H-radiolabeled total inositol phosphates (inositol 1-phosphate, inositol 1,4-phosphate, and inositol 1,4,5-phosphate) following stimulation of cells with 1 Amol/L GnRH-I in the presence of 10 mmol/L lithium chloride. Representative data of assays done in triplicate. B, time courses for turnover of 3H-radiolabeled total inositol phosphates. Cells were stimulated with 1 Amol/L GnRH-I for 90 min in the presence of 10 mmol/L LiCl. Then the medium was replaced with GnRH-I-and LiCl-free medium (dotted line) before assay. Representative data for assays done in triplicate.

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Figure 4. A, qualitative Western blotting showing effects of GnRH receptor activation on phosphorylation of ERK1/2 in cell lines stably transfected with rat GnRH receptor cDNA. Cells were treated with 100 nmol/L 6 D-Trp -GnRH-I for the stated periods of time. Note that B35clones such as B35-2b show variable decreases in the level of phosphorylated ERK1/2 (pERK1/2) following 10-min stimulation whereas the other cell clones exhibit transient increases in ERK1/2 phosphorylation. h-Actin serves as a sample loading control. B, quantitative Western blotting analysis of effects of GnRH receptor 6 activation (via 100 nmol/L D-Trp -GnRH-I) on phosphorylation of ERK1/2 or p38. Data from three independent analyses were quantified and are expressed as fold increase in band intensity standardized according to the level of total ERK1/2 protein. C, Western blot analysis examining changes in the abundance of selected proteins during treatment of cells with 6 100 nmol/L D-Trp -GnRH-I. In HEK293[SCL60] cells (top), there is transient activation of ERK1/2 and p38 and accumulation of phosphorylated FADD, cleaved caspase-9 (c-caspase-9), and cleaved PARP (c-PARP; 4-fold elevation at 72–96 h of treatment in this instance). There is transient dephosphorylation of ERK1/2 but no evidence for rapid activation of p38 in B35-2b cells (bottom). Accumulation of cleaved PARP is less marked in these cells. h-Actin serves as a loading control.

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6 Table 1. Effects of exogenous agents on in vitro cell growth inhibition elicited by 100 nmol/L D-Trp -GnRH-I

Agent Concentration HEK293[SCL60] cells

Ca2+ chelator 5 Amol/L BAPTA/AM Additive* IP3 Ca2+ channel blocker 75 Amol/L 2-APB No effect MEK inhibitor 18 Amol/L PD98059 Partial rescue (8%) P38 inhibitor 10 Amol/L SB203580 No effect PKC inhibitor 100 nmol/L Ro 32-0432 Complete rescue (100%)* PKC inhibitor 0.5 Amol/L Rottlerin No effect cAMP 20 Amol/L 8-Br-cAMP Additive* Caspase inhibitor 20 Amol/L Q-VD-OPh Partial rescue (40%)* Ceramide synthesis block 0.3 Amol/L Fumonisin B1 No effect Fas ligand antagonist 300 Amol/L Kp7-6 No effect

Agent Concentration B35-2 cells

Ca2+ chelator 5 Amol/L BAPTA/AM Additive* IP3 Ca2+ channel blocker 75 Amol/L 2-APB No effect MEK inhibitor 18 Amol/L PD98059 No effect P38 inhibitor 10 Amol/L SB203580 No effect PKC inhibitor 100 nmol/L Ro 32-0432 No effect PKC inhibitor 0.5 Amol/L Rottlerin No effect cAMP 20 Amol/L 8-Br-cAMP Additive* Caspase inhibitor 20 Amol/L Q-VD-OPh Partial rescue (40%)* Ceramide synthesis block 0.3 Amol/L Fumonisin B1 No effect Fas ligand antagonist 300 Amol/L Kp7-6 No effect

NOTE: Cells were treated with agents and growth was monitored at daily intervals over a period of 4 d. Where percentage figures are quoted, these refer to the effect on growth at day 4. Abbreviations: MEK, MAP kinase/ERK kinase; IP3, inositol 1,4,5-trisphosphate. *Represents statistically significant effects where P < 0.002; all analyses were done in triplicate.

2+ the extent of growth inhibition, being more marked in HEK293[SCL60] Inhibition of cellular Ca elevation using 2-APB had no effect on cells (Fig. 4C). Only small elevations in the levels of cleaved GnRH-mediated cell growth inhibition in the cells studied here caspase-9 (upstream to cleaved caspase-3 and cleaved PARP, and a (Table 1). However, phospholipase Ch activity also influences a marker of the intrinsic apoptosis pathway) and phosphorylated range of proteins, including Rac1, a small GTPase involved in FADD (a marker of death-receptor signaling via the extrinsic regulation of apoptosis (45). In HEK293[SCL60] cells, Rac mediates apoptosis pathway) were detectable in HEK293[SCL60] cells. Cleaved GnRH-stimulated cytoskeletal reorganization (46), which may caspase-9 was detectable within 24 hours of treatment. potentially disrupt the highly coordinated cytoskeletal rearrange- Although the level of GnRH receptor expressed in other ments required for cell division. In this respect, it is interesting that transfected cells was low (SKOV3-4, EFO21-1, and HepG2-3), it a proportion of HEK293[SCL60] cells become blocked at the G2 phase was possible to detect low levels of inositol phosphate elevation of the cell cycle after treatment with GnRH (28). Arrest at the G2-M and transient activation of ERK in these cells following treatment checkpoint can trigger apoptosis. Elevation in the level of with GnRH. However, the signaling response was weaker (see Fig. 4 phosphorylated p38 is a recognized marker of G2-M checkpoint for example) and was insufficient to elicit growth inhibition (Fig. 2). stress. We observed activation of p38 in HEK293[SCL60] and B35-2b In SKOV3-4 cells, inositol phosphate production was significant- cells 48 to 96 hours after treatment with triptorelin (Fig. 4). ly stimulated by phospholipase C activator m-3m3FBS (60 Amol/L) Among numerous target proteins, PKCa can activate PKCy and but could be only slightly enhanced by treatment with triptorelin phospholipase D1. PKCy can activate apoptosis via stimulation of (data not shown). Here, m-3m3FBS treatment was also accompa- sphingomyelinase-mediated ceramide production and alteration nied by cell growth inhibition. of mitochondrial membrane lipid composition (47). However, we The in vitro growth-inhibitory effects of triptorelin could be found that rottlerin, an inhibitor of PKCy, did not affect GnRH- partially prevented by the caspase inhibitor Q-VD-OPh (20 Amol/L; mediated growth inhibition (Table 1); neither did fumonisin B1, Fig. 5). Furthermore, a marked cell type–specific rescue was possible an inhibitor of sphingosine N-acyltransferase and de novo with the protein kinase C inhibitor Ro32-0432 (in HEK293[SCL60] cells ceramide biosynthesis. More investigations are required to but not B35 clones; Fig. 5). This is a novel finding, but it is important determine the mechanism of PKC signaling during GnRH to note that Ro32-0432 may inhibit other classes of protein kinases treatment. in addition to PKCa (44) and more work is required to elucidate the Exogenous 8-Br-cAMP acted in an additive fashion with tri- reasons for this cell-specific effect. In contrast, the MAP kinase ptorelin to inhibit in vitro cell growth (Table 1). This suggests that inhibitors PD98059 and SB203580 were unable to rescue cells from GnRH receptor signaling through Gas and adenylate cyclase may be growth inhibition at the doses used in this study (Fig. 5). contextually different from the effects of exogenous 8-Br-cAMP. www.aacrjournals.org 6337 Cancer Res 2008; 68: (15). August 1, 2008

Figure 5. A, results of in vitro cell growth assay indicating that the general caspase inhibitor Q-VD-OPh (20 Amol/L) elicits partial rescue of HEK293[SCL60] or 6 B35-2b cells from growth inhibition by 100 nmol/L D-Trp -GnRH-I. B35-20 or B35-2 cells were not significantly rescued by Q-VD-OPh. Cell growth was measured following 4 d of treatment. Representative data for assays done in triplicate. B, results of in vitro cell growth assay indicating that the protein kinase C inhibitor 6 Ro32-0432 (100 nmol/L) rescues HEK293[SCL60] cells from growth inhibition by 100 nmol/L D-Trp -GnRH-I (top). Cell growth was measured on days 0, 2, 3, and 4 following treatment. Representative data for assays done in triplicate. Western blotting showed that increased levels of cleaved PARP following treatment with 6 100 nmol/L D-Trp -GnRH-I are inhibited by 100 nmol/L Ro32-0432 (bottom). h-Actin serves as a loading control. C, results of in vitro cell growth assay indicating that 6 18 Amol/L PD98059 or 20 Amol/L SB203580 does not rescue HEK293[SCL60] cells from growth inhibition by 100 nmol/L D-Trp -GnRH-I (top), although these agents do prevent rapid activation of p38 or ERK1/2 respectively (bottom). Ro32-0432 at 100 nmol/L prevents ERK1/2 activation, but not p38 activation. h-Actin serves as a loading control. D, results of in vitro cell growth assay indicating that different doses of R032-0432 (90 nmol/L or 180 nmol/L) do not rescue B35cell clones from 6 growth inhibition by 100 nmol/L D-Trp -GnRH-I. Representative data for assays done in triplicate.

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6 Figure 6. In vivo effects of triptorelin (D-Trp -GnRH-I) on tumor xenograft growth. HEK293[SCL60] cell tumor xenografts regressed following treatment for 10 d whereas B35-2b and B35-2 tumor xenografts were growth retarded. Growth of SKOV3-4 xenografts was not affected by treatment with triptorelin. Effects on tumor growth correlate with levels of GnRH receptor expression shown in Fig. 1.

Previous data obtained using pertussis toxin to inhibit Gai may be by a combination of direct and indirect actions on proliferating misleading in view of the recent discovery that the toxin is also an cells. Blood sex steroid concentrations are decreased following inhibitor of acid sphingomyelinase and ceramide production (48). systemic administration of GnRH analogue. Growth of steroid- Differences in the level of GnRH receptor expression also dependent cells is therefore indirectly inhibited. It would be correlated with the extent of tumor xenograft growth inhibition informative to study cells proven to be steroid hormone dependent in vivo (Fig. 6). Growth of HEK293[SCL60] cell tumors expressing using our approach to determine whether steroid hormone receptor high levels of receptor was completely inhibited (Fig. 6). This is function is modified (e.g., by posttranslational modification) by a remarkable effect. GnRH signaling. Likewise influences of GnRH receptor signaling on B35-2b cell tumors expressing a moderate level of GnRH tumor growth via effects on angiogenesis or activity of the innate receptor were more marginally growth inhibited by daily immune system need to be addressed in further investigations. administration of triptorelin than B35-2 tumors, which express Nevertheless, HEK293[SCL60] and transfected B35 cell lines represent lower receptor levels (Figs. 1 and 6). The smaller effect on tumor useful benchmarks to make further comparisons with cells from growth inhibition obtained with transfected B35-2 cell xenografts human tumors that endogenously express GnRH receptor. suggests that 20% in vitro growth inhibition (Fig. 2) translates into poor tumor growth retardation in vivo (Fig. 6). Disclosure of Potential Conflicts of Interest SKOV3-4 cells expressing low levels of GnRH receptor were not in vitro in vivo The authors have no conflicts of interest to declare concerning the contents of this growth inhibited or by triptorelin. Because low research article. levels of GnRH receptor expression do elicit intracellular signaling in SKOV3-4 (and EFO21-1) cells (Fig. 4), attempts to manipulate Acknowledgments this signaling toward stimulation of cell growth inhibition may be Received 1/16/2008; revised 5/27/2008; accepted 5/27/2008. one way to improve the effectiveness of GnRH treatment. How this Grant support: Medical Research Council and Cancer Research UK. may be achieved is the focus of ongoing investigation. The costs of publication of this article were defrayed in part by the payment of page The tumor xenograft experiments complemented our in vitro charges. This article must therefore be hereby marked advertisement in accordance in vitro with 18 U.S.C. Section 1734 solely to indicate this fact. analyses, enabling confirmation of data. However, in many We thank Vicky Warrender and Donald Wilson for cell culture assistance and Ted circumstances the in vivo effects of GnRH are likely to be mediated Pinner and Ronnie Grant for help with the graphics. www.aacrjournals.org 6339 Cancer Res 2008; 68: (15). August 1, 2008

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