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Section of Pediatric Endocrinology/Diabetology, Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, and the James Whitcomb Riley Hospital for Children, Indianapolis, Indiana 46202
Address all correspondence and requests for reprints to: Rosemary Steinmetz, Ph.D., Riley Hospital, Room 2627, 702 Barnhill Drive, Indianapolis, Indiana 46202. E-mail: rsteinme{at}iupui.edu.
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ABSTRACT |
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INTRODUCTION |
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The World Health Organization Classification of ovarian tumors defines several types of ovarian tumors with the most common being derived from the ovarian epithelium, which accounts for approximately 90% of all ovarian malignancies. The second major class of ovarian tumors is designated as sex-cord tumors of the ovary and includes granulosa cell tumors (GCTs), thecomas, and Sertoli-Leydig cell tumors. Of these, GCTs are the most common and are further divided into two distinct subtypes, adult and juvenile, with the latter being found predominantly in females less than 20 yr of age. GCTs, although not as common as epithelial ovarian tumors, are clinically relevant due to their high rate of recurrence and malignant potential (4, 5, 6).
Multiple factors have been shown to be involved in the development of epithelial ovarian cancer, including hormonal input, stimulation by growth factors, gene mutations, inherited risk factors, and environmental factors (7, 8). In contrast, the molecular mechanisms that initiate and support granulosa cell tumorigenesis are not as well defined (5, 6, 9). Prior studies have proposed a role for the FSH receptor system in GCT progression and whereas gonadotropin excess in patients undergoing fertility-enhancing procedures (10, 11) is associated with GCT development, we and others have found little to support the hypotheses that activating mutations of the FSH receptor or mutations in the Gs
subunit are involved (12, 13, 14, 15, 16, 17). Recently, our laboratory reported that G protein-coupled receptor kinase expression is altered in a GCT cell line and in primary tumor samples as compared with nonmalignant granulosa cells (nGC) (18). However, whether these differences result from, or contribute to, tumor cell proliferation is yet to be determined.
Other factors associated with GCTs include alterations in gene expression for p53 (19), Mullerian inhibiting substance (20), and inhibin (19, 21, 22, 23), as well as in the expression of protooncogenes such as c-erbB2, and c-myc (24). To date, no clear relationship between these factors and the development of GCTs has been established. The LH receptor system also has been shown to be strongly associated with the development of GCTs. LH supports the growth of ovarian cancer cells (25, 26) and in studies using LH transgenic mice, GCTs were shown to develop at 100% penetrance in adult females (27). This is a strain-specific phenomenon that is controlled by multiple complex genetic traits (28) and may be related to the formation of tumors in older women. However, this does not explain GCTs that form in young women who do not have elevated LH levels. Thus, is likely that other factors contribute to GCT formation and progression.
The ERK pathway plays a critical role in cell proliferation and survival and represents a conserved signaling system throughout evolution. ERK1 and ERK2 kinases (ERK1/2) are activated via a cascade involving ras/raf/mek proteins (29) and are deactivated through the actions of the cellular phosphatases, MAPK phosphatase 1, 2, and 3 (MKP-1, MKP-2, and MKP-3) (30, 31, 32). Activation of the pathway is normally initiated by the stimulation of growth factors, integrins, or through the activation of G protein-coupled receptor systems (33). Recently, constitutive activation of the ERK1/2 pathway has been documented in leukemia (34, 35), renal cell carcinoma (36), breast cancer (37), and metastatic esophagogastric cancer (38) and in several ovarian cancer cell lines (39).
In the present study, we further examined the prevalence of constitutively active ERK1/2 in ovarian cancer and the mechanisms involved in this activation. Using RNA interference (RNAi), we assessed the consequence of silencing the ERK pathway on tumor cell proliferation. For these studies, we examined HeyC2 and SKOV3 cells, which were derived from epithelial ovarian cancers (40). Additionally, to begin to identify possible contributions of this pathway to the pathogenesis of GCTs, we examined KGN cells that were derived from a recurrent human granulosa cell tumor (41).
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RESULTS |
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). In KGN cells, ERK phosphorylation was 5- to 6-fold higher than in normal granulosa cells (Fig. 1B). In HeyC2 and SKOV3 cells, levels of phosphorylated ERK1/2 were 30- and 20-fold higher, respectively, as compared with the NOSE cell samples in which ERK1/2 activation was consistently found to be low or absent (Fig. 1B). The high basal ERK activity seen in the nGC cells, as compared with the NOSE cells, is likely due to stimulation by supraphysiological levels of gonadotropins, used for oocyte retrieval (42, 43, 44).
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). BRAF, which has only recently been implicated in a variety of cancers, was found to be overexpressed in the cancer cells (Fig. 2B), as compared with the NOSE cells. Phosphorylated MEK1/2 was detected in HeyC2 and KGN cells, but not in SKOV3 cells (Fig. 2C). Phosphorylated MEK1/2 was also detected in the NOSE cells, even though levels of phosphorylated ERK1/2 were low or absent in these samples. Furthermore, because HeyC2 and KGN were found to express phospho-MEK, we tested the effects of the MEK inhibitors, PD98059 and U0126, in these cells. PD98059 (40 µM) decreased phosphorylated ERK1/2 levels modestly, whereas, U0126 (20 µM) decreased ERK phosphorylation dramatically in both cell types (Fig. 3). Collectively, these findings suggest that ERK1/2 phosphorylation may not result from activation by c-raf, but may be induced by BRAF. Additionally, MEK-dependent ERK phosphorylation is present in HeyC2 and KGN cells, whereas in SKOV3 cells, ERK1/2 appear to be activated in a MEK-independent manner. However, studies that measure the individual activities of c-raf and BRAF will be needed to fully understand their respective roles in regulating ERK1/2 activation in ovarian cancer. The difference in the effects of PD98059 and U0126 in HeyC2 and KGN cells is likely due to the specificity and mechanism of action of these two compounds, in that PD98059 inhibits MEK phosphorylation whereas the U0126, which is more specific, blocks the catalytic activity of MEK1/2.
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. Kinase activity was significantly higher in both the HeyC2 and KGN cells, 10- and 20-fold, respectively, as compared with control cells (Fig. 4).
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, top panel) and slightly increased ERK1/2 phosphorylation in HeyC2 cells. In contrast, MDA-MB-231 breast cancer cells, which served as controls and which also express high endogenous ERK1/2 phosphorylation, did respond to SB203580 treatment. HeyC2 and KGN cells also failed to respond to LY294002, whereas LY294002 completely decreased ERK1/2 phosphorylation in the MDA-MB-321 cells (Fig. 5, middle panel). To inhibit PKC, cells were incubated with phorbol 12-myristate 13- acetate (PMA) for 18 h; after this treatment, no significant effect on ERK phosphorylation was seen in any of the cells (Fig. 5, bottom panel).
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). The relatively high level of MKP-1 seen in the NOSE cells is likely to explain the low or absent ERK1/2 phosphorylation seen in these samples even though activated MEK1/2 was detected. MKP-1 levels in the NOSE cells may be sufficient to dephosphorylate endogenous levels of activated ERK1/2. MKP-2 expression was detected in the HeyC2 but not in the KGN cells.
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). In contrast, MKP-2 expression was not altered. The increase in MKP-1 expression was correlated with a decrease in ERK1/2 phosphorylation in HeyC2 and KGN cells, suggesting that low expression of MKP-1 may contribute to the sustained ERK activation seen in both cell types. To confirm that treatment of the cells with ZLLF-CHO specifically impairs proteasome processing, blots were probed with an antibody that recognizes ubiquinated proteins. Results of these studies are shown in the bottom two panels of Fig. 7. ZLLF-CHO increased high molecular weight polyubiquinated proteins, which appear as a smear, as compared with untreated control cells.
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Two pools of siRNAs were designed, each containing four 19-nucleotide oligos specific for ERK1 or ERK2 genes (Table 1). For time course studies, cells were transiently cotransfected with 50 nM of each of the siRNA pools (determined in pilot dose-response studies) and incubated, as before, in maintenance media. Experimental groups consisted of untransfected cells (UT), cells transfected with pools of scrambled siRNA (SC), or cells treated with oligofectamine alone (Oligo). In HeyC2 cells, total ERK1/2 protein expression was decreased by 24 h after transfection and was at its nadir by 72 h (Fig. 8). In KGN cells, the level of ERK silencing was more pronounced: total ERK1/2 expression dropped markedly by 24 h and was undetectable after 2 d (Fig. 8). By 4 d, total ERK1/2 expression began to increase in HeyC2 cells, indicating a loss of the siRNAs from the cells. In contrast, total ERK1/2 expression was still suppressed in KGN cells after 5 d (Fig. 8). In the cells treated with the scrambled siRNAs (control for siRNA specificity), no decrease in total ERK1/2 protein expression was detected. Furthermore, ß-actin expression was similar in all treatment groups. When we examined the effect of ERK1/2 siRNAS on tumor cell proliferation, we found that silencing ERK1/2 gene expression suppressed proliferation for up to 4 d in HeyC2 and 6 d in KGN cells (Fig. 9, A and B). We also examined the effect of ERK knockdown on BrdU incorporation in HeyC2 and KGN cells and found that BrdU incorporation in the siRNA-treated cells decreased to less than 10% of that seen in untreated cells (data not shown).
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). HeyC2 and KGN cells were treated for 5 d with each of these compounds, and methyl thiazolyl blue tetrazolium bromide (MTT) assays were performed at 0, 1, 3, and 5 d. In HeyC2 cells, PD98059 modestly decreased cell proliferation whereas U0126 decreased proliferation by approximately 90% after 5 d. In KGN cells, both PD98059 and U0126 were only partially effective at decreasing cell proliferation. Thus, whereas pharmacological down-regulation of the ERK pathway is effective at suppressing tumor cell growth in vitro in HeyC2 cells, in KGN cells, MEK inhibitors were less effective. In contrast, silencing of the pathway using RNAi was effective in both cell types.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Multiple factors are associated with the constitutive activation of the ERK1/2 pathway (34, 35, 36, 37), including MEK-dependent and -independent mechanisms (38, 52). In HeyC2 and KGN cells, PD98059 partially deactivated ERK1/2, whereas U0126 completely decreased ERK phosphorylation, strongly suggesting that MEK-dependent mechanisms are involved. Additionally, because we were unable to detect phosphorylation of the S338 residue, we further hypothesize that c-raf is likely not to be required for constitutive ERK1/2 activation in their tumor cells. Barry et al. (38) also found that c-raf expression was absent in bone metastases of esophagogastric cancer and also concluded that the lack of phosphorylation of this key serine residue likely denotes c-raf-independent ERK1/2 activation in their tumor cells. Activating mutations in the BRAF gene have recently been shown to be associated with malignant melanomas (53, 54, 55), colon (56), thyroid (57, 58), and serous ovarian (56, 59) cancers. We are currently examining the BRAF sequence in our cancer cells to determine whether mutations are present and whether BRAF activity is increased either as a result of such a mutation or as a result of overexpression.
We found no dependence of ERK1/2 activation on input from the p38 MAPK, PI3K, or PKC pathways. The small increase in ERK phosphorylation seen with SB203580 treatment is likely a nonspecific effect mediated through inhibition of the PDK-1 or AKT pathways. PKC plays an important role in ligand-stimulated sustained ERK1/2 activation (60, 61); thus, our finding that down-regulation of PKC does not influence sustained ERK activation in the ovarian cancer cells supports the hypothesis that ERK1/2 activation is constitutive in manner.
A gain-of-function mutation that results in enhanced ERK1/2 signaling capabilities, similar to that reported in Drosophila (62), could also support ERK1/2 activation in the cancer cells. Using sequence analysis, two polymorphisms (C995T and T1041C) were detected in the ERK2 gene sequence in the KGN cells (data not shown). These polymorphisms, however, do not result in changes in the amino acid sequence and are unlikely to contribute to the observed activation.
Cell-modeling studies have shown that MKP activity can modulate the steady-state levels of phosphorylated ERK1/2 (61, 63), such that when MKP activity is low, MAPK dephosphorylation is impaired, resulting in high ERK1/2 signaling activity. Low levels of MKP-1 expression were seen in both HeyC2 and KGN cells as compared with the NOSE cells, whereas MKP-2 expression was detected only in HeyC2 cells. Furthermore, treatment of the cells with ZLLF-CHO increased MKP-1 expression and decreased ERK1/2 phosphorylation, supporting the hypothesis that down-regulation of MKP-1 expression contributes to ERK1/2 constitutive activation in these cells. These findings are supported by recent studies showing that sustained ERK1/2 activation is the result of MKP-1 degradation through the ubiquitin/proteasome pathway and that overexpression of MKP-1 directly decreases ERK1/2 phosphorylation (64). Additionally, in studies examining mechanisms regulating proteasome inhibitor- mediated apoptosis, increased MKP-1 expression and decreased ERK1/2 phosphorylation were seen after treatment of cells with ZLLF-CHO (51). We do not know what role MKP-2 plays in ERK activation in these cancer cells. In HeyC2 cells, ERK1/2 are constitutively activated even though MKP-2 is highly expressed. Furthermore, we were unable to detect an increase in MKP-2 expression after exposure of the cells to ZLLF-CHO in either cell type. These findings could mean that MKP activity is impaired in HeyC2 cells and severely down-regulated in KGN cells, or that MKP-2 does not have a significant role in regulating ERK1/2 dephosphorylation in these cells. Orlowski et al. (51) also detected high endogenous levels of MKP-2 in breast cancer cells and whereas treatment of these cells with ZLLF-CHO did induce MKP-2 accumulation and decrease ERK1/2 phosphorylation, these changes were not significant. Thus, they also hypothesized that MKP-2 is not a major component of the negative feedback loop that governs ERK1/2 desphosphorylation. However, it is possible that this is specific to cancer cells and does not mimic MKP-2 expression or activity in normal cells.
This is the first study to use RNAi to suppress ERK1/2 protein expression and evaluate the effect of such intervention on ovarian tumor cell proliferation. Treatment of the cells with ERK1/2 siRNAs dramatically suppressed tumor cell proliferation, suggesting that ERK1/2 activation plays a major role in tumorigenesis. We speculate that silencing of this pathway not only suppresses cell proliferation but may also suppress tumor cell metastases by inhibiting the expression of tissue proteases involved in this process. Work is ongoing to determine the effect of ERK siRNAs on apoptosis and whether there is a differential effect of this treatment in malignant as compared with normal ovarian cell types.
GCTs are a clinically relevant form of ovarian cancer in that they affect both women and young girls, have a significant malignant potential, and require decades of follow-up care due to their high rate of recurrence (65). In an examination of a small panel of human GCTs, we detected high levels of ERK1/2 phosphorylation in 75% of the metastatic tumors examined (data not shown). This, coupled with the fact that the KGN cells used in the current studies were derived from an aggressive recurring GCT, suggests that constitutive activation of the ERK pathway may be required for GCT recurrence. In summary, these studies provide a significant step in understanding key intracellular mechanisms involved in the progression of both GCTs and epithelial ovarian tumors and may facilitate the development of targeted forms of treatment for these cancers.
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MATERIALS AND METHODS |
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Cell Culture
KGN cells, a previously characterized human granulosa tumor cell line, were a gift from Drs. Y. Nishi, and T. Yanase (Kyushu University, Fukuoka, Japan). Cells were maintained for all experiments in DMEM/F12 media supplemented with 10% FBS, and 1% penicillin-streptomycin. HeyC2 and SKOV3 ovarian and MDA-MB-231 breast cancer cells were generous gifts from Dr. R. M. Bigsby (Indiana University School of Medicine, Indianapolis, IN). HeyC2 cells were maintained in MEM supplemented with 5% FBS, 1% HEPES, 1% l-glutamine, and 1% NEAA. SKOV3 cells were maintained for all experiments in McCoy’s 5A modified medium supplemented with 10% FBS, 1% HEPES, 1% L-glutamine, and 1% MEM sodium pyruvate. MDA-MB-231 cells were maintained in MEM supplemented with 5% FBS, 1% HEPES, 1% L-glutamine, 1% NEAA, and insulin, 6 ng/ml. All cell lines were incubated at 37 C with 5% CO2.
Human Tissue Samples
All human tissues and cells were collected after signed patient consent. Protocols for tissue and cell collection were approved by the Indiana University Institutional Review Board. Normal ovarian surface epithelial cells extracts were prepared from NOSE cells (a gift from Dr. R. M. Bigsby) harvested from females undergoing complete hysterectomies for nonmalignant conditions. Epithelial cells were collected and maintained in MCDB105:M199 media (1:1) containing 20% FBS, 1% L-glutamine and sodium pyruvate, gentamicin (25 µg/ml final concentration) and EGF (10 ng/ml final concentration).
Human nGC were harvested from follicular fluid collected during oocyte retrieval for in vitro fertilization at the Clarian Health Reproductive Biology Laboratory. Follicular fluid was collected and granulosa cells were isolated as previously described (66). Essentially, follicular fluid was centrifuged at 750 x g over a ficoll gradient with the red blood cells collecting at the bottom of the tube and the granulosa cells partitioning at the interface. Granulosa cells were removed and washed twice in DMEM/Ham’s F12 media, and protein extracts were prepared as above. BCA protein analysis was performed to determine total protein concentration.
Inhibitor Studies
For all inhibition experiments, cell lines were plated at 2 x 105 cells per well in six-well Falcon tissue culture plates. The cells were allowed to attach and recover for 24 h. The following inhibitors were then added to three of the six wells per plate: PD98059 (40 µM), U0126 (20 µM), SB203580 (20 µM), LY294002 (20 µM), for 1 h, or PMA (1 µM) overnight. Three untreated wells per plate served as controls. For proteasome inhibition, cells were incubated with 0 or 5 µM of the inhibitor ZLLF-CHO [(Benzyloxycarbonyl)-Leu-Leu-phenylalaninal] (Calbiochem) for 6 h at which time total proteins were extracted, as above, for Western analysis. Blots were then stripped and reprobed to detect the presence of polyubiquinated proteins.
For proliferation studies using U0126 and PD98059, cells were plated at 10 x 103 cells per well for KGN and 5 x 103 cells per well for HeyC2 cells. Cells were incubated overnight to allow for cell attachment and then treated with either PD98059 (40 µM) or U0126 (20 µM). Cells were retreated every other day. Proliferation was determined at 0, 1, 3, and 5 d.
Western Blot Analysis
Protein lysates were prepared from each of the cell lines and from the in vitro fertilization-derived granulosa cells, using T-PER protein extraction buffer. Protein concentrations for all samples were determined using the BCA protein assay. Lysates were then boiled and loaded into a 10% SDS-polyacrylamide gel. Total protein (30 µg) was analyzed for MKP-1, MKP-2, and raf isoform expression. All other Western blots were performed using 10 µg total protein per sample. A monoclonal antibody for ß-actin was used to ensure equal loading and for normalization of protein levels. All Western blot analyses were performed following procedures by Laemmli (67). LumiGLO and peroxide chemiluminescent substrates were used to detect signal.
Kinase Activity Assay
Kinase assays that determined the ability of constitutively activated ERK1/2 to phosphorylate the transcription factor ELK-1, a known ERK substrate, were performed following the manufacturer’s protocol. For the assay, 2 x 106 KGN or HeyC2 cells were washed once with cold PBS, and protein extracts were prepared using lysis reagent (20 mM Tris, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1% Triton; 2.5 mM sodium pyrophosphate; 1 mM ß-glycerolphosphate; 1 mM Na3VO4; and 1 µg/ml Leupeptin). Samples were sonicated and centrifuged (10,000 rpm) for 10 min. Protein concentrations were determined using the BCA protein assay reagent. Total protein (200 µg) from each cell line was mixed with 15 µl of immobilized phospho-ERK1/2 MAPK (Thr202/Tyr204) monoclonal antibody that selectively precipitates active (phosphorylated) ERK MAPK. A positive control was made by adding 2 µl of the kinase control (MAPK supplied by the manufacturer) to a second 200-µg protein aliquot from cells that do not constitutively express phosphorylated ERK. This positive control was treated identically to the other samples. All samples were incubated overnight at 4 C with gentle rocking. Immobilized phosphorylated ERK proteins were eluted from the beads and reacted with an ELK-1 fusion protein substrate in the presence of ATP and kinase buffer (25 mM Tris, pH 7.5; 5 mM ß-glycerolphosphate; 2 mM dithiothreitol; 0.1 mM Na3VO4; 10 mM MgCl2). Western blotting was performed using an antibody that specifically reacts with ELK-1 that is phosphorylated on Serine 383, a requirement for ELK-1-induced transcription. The active phosphorylated ELK-1 signal was detected using the LumiGLO chemiluminescent substrates. Blots were scanned and quantified by densitometry.
RNA Interference (RNAi)
For total ERK protein knockdown and cell proliferation experiments, cells were plated in six-well tissue culture dishes in their respective maintenance media and incubated for 24–48 h before transfection. On the day of transfection, media were replaced with serum-free and antibiotic-free Optimem (Invitrogen Life Technologies) and transient transfections were performed using 50 nM of each ERK SMARTpool (Dharmacon RNA Technologies, Lafayette, CO) complexed with oligofectamine (Invitrogen Life Technolgies) for 5 h at 37 C. Each pool consists of four 19-nucleotide sequences each specific and complementary to ERK1 or ERK2 mRNA (Table 1
). After the transfection, the media were replaced with serum-containing maintenance media and the cells were incubated for the specified times. Groups of cells were also transfected with a pool of four scrambled siRNA sequences which served as a control for specificity. In each experiment, four to six replicates per dose and time point were analyzed. All experiments were repeated at least three times. After transfection, the media were removed and replaced with the respective serum containing maintenance media for each cell line. Fresh media were added every 2 d for the duration of the experiment.
MTT Assays for Cell Proliferation
Cell proliferation was measured using the methyl thiazolyl blue tetrazolium bromide (MTT, Sigma/Aldrich St. Louis, MO) colorimetric dye assay (68). At each time point, HeyC2 and KGN cells were incubated with 0.3 ml MTT dye (1 mg/ml in serum-free media) for 2 h at 37 C. Color was developed by the addition of 200 µl of 0.04 N HCl in isopropyl alcohol. The color reactions were then transferred to a 96-well plate and the absorbance was monitored at 570 nM.
Statistics
Results are expressed as mean ± SEM. Statistical comparisons were made by using ANOVA with subsequent application of the Scheffe test or t test where appropriate.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: FBS, Fetal bovine serum; GCT, granulosa cell tumor; MEK, MAPK kinase; MKP, MAPK phosphatase; MTT, methyl thiazolyl blue tetrazolium bromide; NEAA, nonessential amino acids; nGC, nonmalignant granulosa cells; NOSE, normal ovarian surface epithelial cells; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RNAi, RNA interference; siRNA, small interfering RNA; ZLLF-CHO, benzyloxycarbonyl-Leu-Leu-phenylalanine.
Received for publication February 26, 2004. Accepted for publication June 30, 2004.
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inhibits luteinized human granulosa cell steroid production in vitro. Am J Obstet Gynecol 172:1505–1510[CrossRef][Medline]
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