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Division of Endocrinology and Metabolism, Endocrine Research Unit (K.R.K., O.I.F., L.K.B., C.A.C.) and Division of Experimental Pathology (M.A.Z., P.C.R.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Dr. Cheryl Conover, Endocrine Research Unit, Mayo Clinic, 200 First Street Southwest, 5-194 Joseph, Rochester, Minnesota 55905. E-mail: conover.cheryl{at}mayo.edu.
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Abstract |
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Introduction |
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Ovarian cancer cells can express varying levels of receptors for peptide, lipid, and steroid growth factors (2). Understanding the differences in receptor expression and function between malignant and normal cells is critical for advancing knowledge of ovarian cancer cell biology, and could lead to the development of tumor specific therapies. We have focused on the function of the IGF system (3). This report investigates the role of the insulin receptor, which is homologous to the IGF-I receptor, in modulating growth of malignant ovarian epithelial cells.
The insulin receptor is a member of the tyrosine kinase growth factor receptor family (4, 5). It is a heterotetrameric structure composed of two 
-subunits and two ß-subunits connected by disulfide bonds. Binding of insulin to the extracellular -subunits initiates activation of the cytoplasmic tyrosine kinase domain in each ß-subunit with subsequent tyrosine phosphorylation of the ß-subunits and activation of intracellular signaling.
Although primarily thought of as mediating the metabolic effects of insulin in insulin target tissues such as adipose tissue and skeletal muscle, the insulin receptor has been shown to mediate a proliferative response in certain transformed cells (6, 7, 8). Indeed, hyperinsulinemia and overexpression of insulin receptors appear to play an important role in the biology of human breast cancer (9, 10, 11, 12, 13). The large majority of human breast cancers have increased levels of functional insulin receptors that are mainly expressed by malignant epithelial cells (10, 11, 14). Insulin receptor content in node-negative breast cancers has been suggested to be predictive of clinical outcome, with tumors expressing very high levels of insulin receptor associated with decreased 5-yr disease-free survival (15). Furthermore, cultured breast cancer cells express high levels of insulin receptor and many, but not all, of these cell lines have been shown to respond to low doses of insulin with increases in DNA and protein synthesis (7, 8). Thus, hyperinsulinism has been considered a contributing factor in breast cancer growth.
In comparison, specific studies on the possible role of insulin and insulin receptors in ovarian cancer are limited. In situ hybridization and immunohistochemical analysis showed ubiquitous expression of the insulin receptor in normal human ovary (16). In 1994, Beck et al. (17) used an RIA to analyze insulin receptor content in extracts of human ovarian tissue specimens, including benign and malignant tumors and normal ovary. They concluded that insulin receptors were present in all samples tested, albeit at variable levels. However, it should be noted that extracts of normal ovary containing primarily nonepithelial cell types may not be appropriate comparisons to cancers arising from the surface epithelium. Nonetheless, it was postulated that hyperinsulinism may be involved in the growth of epithelial ovarian malignancies (17).
Interestingly, there have been recent reports of two insulin receptor isoforms that differentially respond to IGF-II with mitogenic signaling. Insulin receptor isoforms A and B differ in 12 amino acids due to alternative splicing of exon 11 (18). This exon appears to have negative effects on binding of the IGFs but not of insulin (19), so isoform B (containing exon 11) binds only insulin, whereas isoform A (lacking exon 11) binds insulin as well as IGF-II (20). While isoform B mediates the metabolic effects of insulin, isoform A initiates mitogenic signaling cascades (20). The exact physiologic function of insulin receptor isoform A is unclear. However, it has been implicated in fetal development and cancer (20, 21). If IGF-II is present in abundance, then insulin receptor isoform A could contribute to mitogenesis. Thus, it may be relevant that the specific IGF-II receptor responsible for internalization and degradation of this growth factor is down-regulated in a variety of tumors, including epithelial ovarian cancer (22, 23, 24, 25, 26).
In this study we tested three hypotheses: 1) human epithelial ovarian cancer cells have elevated insulin receptor levels relative to normal ovarian surface epithelial cells; 2) insulin receptors on human epithelial ovarian cancer cells mediate a mitogenic effect of insulin; and 3) insulin receptors on human epithelial ovarian cancer cells bind and mediate a mitogenic response to IGF-II.
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Materials and Methods |
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-MEM and fetal bovine serum were from Irvine Scientific (Santa Ana, CA). Medium 199, MCDB 105, and RIA grade BSA were from Sigma (St. Louis, MO). Monoclonal antibodies to the insulin receptor -subunit (47–9 and clone 83–7) were purchased from Neomarkers, Inc. (Freemont, CA). Monoclonal and polyclonal antibodies to the insulin receptor ß-subunit were from Oncogene Research Products (Boston, MA) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. Mouse IgG1 was from Research Diagnostics, Inc. (Flanders, NJ), and fluorescein isothiocyanate (FITC)-labeled antimouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA).
Cell cultures
Epithelial ovarian carcinoma cell lines (OV17, OV167, OV177, OV202, OV207, and OV266) were obtained from primary ovarian tumor specimens and cultured as described (3). Normal ovarian surface epithelial cells (OSE) were derived as described (27) from ovaries of women undergoing oophorectomy for benign gynecologic indications. To enhance their lifespan in culture, uniformly cytokeratin positive cultures were infected with pZipSVtsA58, a retrovirus encoding a temperature-sensitive mutant form of the SV40 large T antigen (28, 29), generating OSE(tsT) lines. At 34 C, the permissive temperature for the SV40 large T antigen, these cells rapidly proliferate. At the restrictive temperature of 39 C the mutant large T antigen is unstable and the cells cease to proliferate; i.e. transient exposure to the large T antigen has not immortalized the cells. Immunohistochemical detection of the SV40 large T antigen shows decreased nuclear staining after 8 h and no expression after 48 h at 39 C (data not shown). All experiments with OSE(tsT) cells were performed after culture at 39 C for 72 h. The breast cancer cell line, MCF-7, was from American Type Culture Collection (Manassas, VA) and cultured in phenol red-free DMEM:F12 supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum.
Binding studies
Binding assays were performed directly on cell monolayers 5–7 d after the last media change as previously described (3, 30). The cells were washed three times with cold HEPES binding buffer plus 0.5% BSA and then incubated for 2.5 h at 15 C with 125I-insulin or 125I-IGF-I (50,000 cpm). [Leu27]IGF-II (100 ng/ml) was included in the 125I-IGF-I binding assay to distinguish type I IGF receptors from cell-associated IGF binding proteins (3, 31). Binding was done at pH 8.0 for insulin and IGF-II and at pH 7.4 for IGF-I. Nonspecific binding was defined as the amount of radioactive insulin or IGF-I bound in the presence of excess unlabeled insulin or IGF-I, respectively. Nonspecific binding was less than 1% of the total counts added and was subtracted from total binding to determine specific binding. For some studies, 47–9, a specific antagonist for the -subunit of the insulin receptor (30, 32), was added 30 min before binding studies were initiated.
Flow cytometry
Ovarian carcinoma cell lines were incubated with mouse IgG1 or clone 83-7 anti-insulin receptor antibodies for 1 h on ice, then washed with PBS/0.1% BSA/0.02% sodium azide (FACS buffer). FITC-labeled secondary antibody was added for 30 min, followed by washing as above. Cells were resuspended in FACS buffer containing 100 ng/ml propidium iodide before being run on a Becton Dickinson and Co. (San Jose, CA) FACScan cytometer in the Mayo Clinic Flow Cytometry/Optical Morphology Resource. Data were analyzed using WinMDI freeware from The Scripps Research Institute after gating on viable cells.
Affinity cross-linking
Ovarian carcinoma cell cultures were washed three times with cold HEPES binding buffer and 0.5% BSA and then incubated with 125I-insulin (3 x 106 cpm) with and without unlabeled insulin (5 nM) and IGF-I (5 nM) at 15 C for 2.5 h. Affinity cross-linking of monolayers was performed with disuccinimidyl suberate (Pierce Chemical Co., Rockford, IL) as described (3, 30). Reduced samples (containing 100 mM dithiothreitol) were electrophoresed using SDS-PAGE (5–15% linear gradient), dried, and exposed to film.
Immunoprecipitation
Cells were cross-linked as above after incubation for 2.5 h with 125I-IGF-II. After quenching, cells were solubilized in buffer containing protease and phosphatase inhibitors (33). Lysates were sonicated and centrifuged at 12,000 rpm for 10 min at 4 C. Cell lysates were precleared with protein G Plus/protein A-agarose beads (Oncogene Science, Inc., Uniondale, NY), and then incubated for 2.5 h at 4 C with mouse IgG1. Protein G/Plus protein-A agarose was included for the final 30 min of the incubation. Beads were pelleted and the supernatants incubated overnight at 4 C with 5 µg insulin receptor ß-subunit monoclonal antibody (Ab-3, Oncogene Science, Inc.), after which beads were added as above to capture immune complexes. The beads were washed five times with lysis buffer before the immunoprecipitated proteins were processed on 5–15% linear acrylamide gradients using SDS-PAGE under reducing conditions, after which gels were dried and exposed to a PhosphorImager (Amersham Biosciences).
3H-thymidine incorporation
Cell monolayers, approximately 75% confluent, were washed and changed to DMEM plus 0.1% BSA (serum-free medium, SFM) for 72 h. Cells were then treated as indicated, with 3H-thymidine (0.5 µCi/well) added for the final 18 h of a 48 h incubation. Radiolabel incorporated into acid-precipitable material was quantitated as described (3).
Cell replication
Triplicate wells at approximately 75% confluency were washed twice and changed to SFM for 24 h. Cells were washed twice again and changed to SFM with the indicated experimental conditions for 72 h. At the end of the incubation period, adherent cells were trypsinized and counted in triplicate with a hemacytometer.
5' nuclease assay
The RNeasy Mini Kit (QIAGEN, Inc., Valencia, CA) was used to prepare total RNA from cell lines. RNA was treated with deoxyribonuclease (DNA-free, Ambion, Inc., Austin, TX) before RT of 400 ng RNA using TaqMan Reverse Transcription Reagents (PE Biosystems, Foster City, CA) according to the manufacturer’s instructions. Absence of genomic DNA contamination was confirmed by PCR (see below) using isolated RNA without RT as template.
The 5' nuclease assays (formerly referred to as real-time quantitative PCR analyses) were performed using the ABI PRISM 7700 Sequence Detection System and software (PE Applied Biosystems). All reagents were from PE Biosystems. Primer and probe sequences for specific amplification and detection of the targets (insulin receptor isoforms A and B) as well as the reference gene (nuclear DNA-encoded gene 28S rRNA) were selected using the Primer Express software from the published sequences of each gene, and are as follows: insulin receptor isoform A forward primer 5'-CTGCACAACGTGGTTTTCGT-3', reverse primer 5'-ACGGCCACCGTCACATTC-3', and probe 5'-CCCAGGCCATCTCGGAAACGC; insulin receptor isoform B forward primer 5'-CGTCCCCAGAAAAACCTCTTC-3', reverse primer 5'-ACGGCCACCGTCACATTC-3', and probe 5'-CCGAGGACCCTAGGCCATCTCGG-3'. Insulin receptor probes were supplied labeled at the 5' end with the fluorescent reporter dye 6-FAM (6'carboxyfluorescein) and labeled at the 3' end with the quencher dye TAMRA (6'carboxytetramethylrhodamine) and were phosphate-blocked at the 3' end to prevent extension. The 28S primers and probe were used as described (34). Triplicate samples were analyzed in comparison with standard curves for each gene. The standard curves were created by performing the 5'-nuclease assay on 101–107 copies of separate DNA templates encoding the genes of interest. These single-stranded oligonucleotides were synthesized, purified using polyacrylamide gel electrophoresis, and quantitated by spectrophotometry by Integrated DNA Technologies, Inc. (Coralville, IA). The standards correspond directly to the amplicon for each gene, and are therefore 80 nucleotides in length for isoform A, 99 nucleotides for isoform B, and 85 nucleotides for the 28S standard (sequences available upon request). Comparison of the cycle threshold observed with a specific primer and probe combination in test samples to the linear portion of the gene’s standard curve allowed estimation of copy number for each gene, which was subsequently normalized to the 28S copy number.
Statistical analyses
All values are expressed as the mean ± SE. ANOVA was used when comparing multiple groups. Results were considered significant at P < 0.05.
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Results |
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presents specific 125I-insulin binding to human epithelial ovarian cancer cells in culture. All six of the ovarian cancer cell lines specifically bound insulin. Five of the six cell lines had levels of insulin binding equal to or greater than those in MCF-7 breast cancer cells, a known insulin-responsive cell line (7, 8). OV17 and OV167 had the highest specific insulin binding; OV202 had specific binding comparable with levels seen in four separate OSE(tsT) lines. Except for OV202, the ovarian cancer cells had 2- to 10-fold elevated insulin binding compared with OSE(tsT) cells. Flow cytometric analyses of insulin receptor expression using an antibody that recognizes the extracellular chain confirmed the presence of insulin receptors on five of six ovarian cancer cell lines (Fig. 1). In addition, low levels of insulin receptors were demonstrated on one of two OSE(tsT) cell lines analyzed in this manner, with the other OSE(tsT) line expressing undetectable levels of insulin receptor.
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-subunit of the insulin receptor, 47–9 (32). 47–9 dose-dependently inhibited 125I-insulin binding but not 125I-IGF-I binding to ovarian cancer cells (Fig. 2A), consistent with its specificity for insulin receptors and not IGF-I receptors. At 10 nM, 47–9 inhibited more than 80% of insulin binding and less than 10% of IGF-I binding, indicating that the 125I-insulin is binding to the insulin receptor and not the IGF-I receptor. 47–9 significantly inhibited 125I-insulin binding in the five ovarian cancer cell lines that bound elevated levels of insulin (Fig. 2B).
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). 125I-Insulin binding was performed in the absence or presence of unlabeled insulin and IGF-I. Following binding, cells were cross-linked, solubilized and subjected to SDS-PAGE under reducing conditions. For all of the ovarian cancer cell lines tested (including OV177 and OV266, not shown), there was a single labeled band at approximately 135 kDa, the expected size of the insulin (or IGF-I) receptor chain. Labeling was displaceable with unlabeled insulin with little or no effect of unlabeled IGF-I (each at 5 nM), indicating that 125I-insulin binding occurred predominantly to the -subunit of the insulin receptor (30, 35).
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, OV167, OV177, and OV266 increased 3H-thymidine incorporation in response to incremental doses of insulin. A significant effect was seen at 0.1 nM insulin. This concentration is more than 4 orders of magnitude lower than the concentration of insulin commonly included in tissue culture media (10 µg/ml, or 1.7 µM), which promotes growth by cross-reaction with the IGF-I receptor. Insulin treatment also caused an increase in the percentage of cells in S-phase, the DNA replicative phase of the cell cycle, as assessed by flow cytometry (data not shown). In contrast, there was no apparent effect of insulin on 3H-thymidine incorporation in OV17, OV202, and OV207 even at 100 nM (data not shown). This lack of apparent response to insulin might be due to the high basal rate of DNA synthesis exhibited by these cells. To overcome this technical issue, we performed cell number analyses after treatment with 1 and 10 nM insulin (Fig. 4B). OV17 and OV207 cells responded to insulin treatment with increases in cell number. OV202 cells, while competent to proliferate as determined by their response to IGF-I (3), did not increase in number when treated with insulin (data not shown and Table 2).
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). IGF-II at both 1 nM and 10 nM significantly stimulated 3H-thymidine incorporation in OV177 and OV266 cells and cell proliferation in OV17, OV167, and OV207 cells. OV202 cells, which increase in cell number in response to IGF-I (3), were not increased in cell number after IGF-II treatment. The response of each cell line to the respective dose of insulin was very similar to the response (or lack thereof, in OV202) to IGF-II.
To clarify the receptor responsible for binding the IGF-II in these studies, the ability of unlabeled IGF-II to displace 125I-insulin binding to the insulin receptor was tested (Table 3). Significant displacement was seen with 1 nM IGF-II in OV17, OV177, and OV207 cells. In all cell lines except for OV17, the displacement with 10 nM IGF-II (84–90%) was similar to that obtained with 1 nM insulin (89–96%). Displacement of 125I-insulin binding by unlabeled IGF-II suggests mediation of the mitogenic response of IGF-II, at least in part, through the insulin receptor in these ovarian cancer cell lines.
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). A strong signal representing 125I-IGF-II binding to the chain of the insulin receptor is seen at approximately 135 kDa only in the anti-insulin receptor immunoprecipitates, and this signal is inhibited by inclusion of cold insulin in the binding reaction.
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Discussion |
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Our first hypothesis was that epithelial ovarian carcinoma cell lines had elevated levels of insulin receptor in comparison to normal ovarian epithelium. All six of the ovarian cancer cell lines specifically bound 125I-insulin, albeit at variable levels, and five of six of the lines showed positive staining with a primary antibody recognizing the insulin receptor receptor chain. These cell lines were derived from primary tumors and the variability may relate to the different tumor type, stage, and grade (see Ref. 3), although a larger number of tumor specimens would be required to test this possibility. That insulin was binding to insulin receptors and not to related receptor types such as IGF-I receptors was established by several criteria: 1) specific 125I-insulin binding to cell monolayers; 2) affinity cross-linking of 125I-insulin to the 135-kDa -subunit of the insulin receptor that could be blocked by unlabeled insulin and not by IGF-I; and 3) inhibition of 125I-insulin, but not 125I-IGF-I, binding by a selective insulin receptor blocking antibody, 47–9.
Insulin binding analyses of both malignant and untransformed ovarian epithelial cells (Table 1) support the hypothesis that human epithelial ovarian cancer cells have elevated insulin receptor levels relative to normal ovarian surface epithelial cells. Five of the six ovarian cancer cell lines exhibited 2- to 10-fold higher specific 125I-insulin binding than the four OSE(tsT) cell lines. Binding studies were done after incubation of OSE(tsT) cells at 39 C, the temperature at which the mutant SV40 large T antigen is dysfunctional. However, expression of SV40 large T antigen during expansion at 34 C may have induced insulin receptor expression (38). If so, elevated levels of insulin receptors may still be present during the binding assays, meaning that there would be an even more striking difference between truly normal and malignant ovarian epithelial cells in terms of insulin receptor expression than demonstrated in these experiments. In fact, OSE(tsT)-130 cells held at 34 C have low levels of insulin receptor expression that decrease after 72 h at 39 C, whereas OSE(tsT)-14 cells have similar levels at each temperature (data not shown).
Normally, epithelial cells and fibroblasts express low levels of insulin receptor. While a consistent correlation between levels of growth factor receptor expression and the presence or magnitude of a positive response to the growth factor cannot be assumed in any system, it has been shown experimentally that overexpression of insulin receptors can increase responses to insulin. For example, an increased mitogenic response to low levels of insulin has been demonstrated in murine fibroblasts transfected with human insulin receptor (39). Additionally, overexpression of insulin receptors has been shown to lead to insulin-dependent transformation (40).
Our study supports the second hypothesis that insulin receptors on epithelial ovarian cancer cells mediate a mitogenic effect of insulin. All of our epithelial ovarian cancer cell lines except OV202, which had the lowest specific insulin binding, responded to low levels of insulin with an increased growth response. There are several possible underlying mechanisms that could account for a mitogenic effect of insulin. Insulin could be signaling through a hybrid insulin/IGF-I receptor (41, 42, 43). Ovarian cancer cells express IGF-I receptors (3), so such hybrids probably exist on these cells. However, hybrid receptors preferentially bind IGF-I and not insulin (42, 43), making it unlikely that hybrids are mediating proliferative responses to insulin. A second possibility is that insulin is binding to an atypical IGF-I receptor, which binds both IGF-I and insulin with high affinity (44, 45). If this were the case, however, insulin binding and action would not be inhibited with the specific insulin receptor antibody, 47–9. A third possibility, and the one our data favor, is that insulin is signaling through the insulin receptor. Furthermore, the ability of unlabeled IGF-II to compete for 125I-insulin binding to the insulin receptor and the mitogenic response of the cell lines to insulin implicate insulin receptor isoform A as mediating these effects.
Insulin receptor isoform A (lacking exon 11) is preferentially expressed in fetal tissues as well as in certain breast and colon cancers (20, 21), initiates mitogenic responses, and binds both insulin and IGF-II (20). Our data showing that IGF-II can displace 125I-insulin binding to the ovarian cancer cells are consistent with isoform A expression. In addition, results from the 5' nuclease assay indicate that ovarian carcinoma cell lines contain more mRNA for isoform A than for isoform B. Analysis of mRNA from normal human liver, a tissue in which isoform B predominates (18), confirmed that preferential detection of isoform A was specific to the samples studied and not due to differential assay sensitivity (data not shown). However, further analysis of the isoform distribution in normal OSE cultures (rather than the SV40-expressing OSE) will be required to determine whether a high isoform A to B ratio is characteristic of ovarian epithelial cells in general or a hallmark of ovarian malignancy.
Our findings support the third hypothesis that insulin receptors on epithelial ovarian cancer cells can mediate a mitogenic effect of IGF-II. The IGF-II receptor does not have signaling potential and it was previously believed that most, if not all, of the biological effects of IGF-II were mediated through the IGF-I receptor (46). It was only recent genetic data that revealed a role of the insulin receptor in mediating the effect of IGF-II in fetal growth in mice and in IGF-I receptor-null cells (36, 37). IGF-II was as effective as insulin in stimulating a proliferative response in all cell lines except OV202 (Table 3). IGF-II was able to compete for tracer levels of radiolabeled insulin binding to the insulin receptor, and IGF-II binding was directly visualized after cross-linking and immunoprecipitation of the insulin receptor. It is significant that OV202 cells that are capable of proliferating in response to IGF-I (3) are unable to proliferate in response to insulin or IGF-II. This provides indirect evidence that the low doses of insulin and IGF-II used in these in vitro studies, as predicted based on ligand binding affinities, are indeed activating insulin receptors. These studies were not intended to exclude participation of the IGF-I receptor in the mitogenic response to IGF-II. Nonetheless, the data provide strong supportive evidence that the insulin receptor may also play an important role in the response to local IGF-II.
The possible clinical significance of elevated insulin receptors in ovarian cancer is unknown. Insulin receptor overexpression may confer selective growth advantage in clinical syndromes associated with hyperinsulinemia. Indeed, hyperinsulinism has been suggested as an underlying cause of breast cancer (11, 12, 13). Although equivalent epidemiological studies on the relationship between such factors as insulin, nutrition, and ovarian cancer are limited (47), the observation that ovarian cancer varies in incidence with geographical location (1) indicates that environmental or cultural influences could contribute to the risk of developing ovarian cancer.
While hyperinsulinism has no clear connection to ovarian cancer, the IGF-II receptor appears to be down-regulated (26) in a certain percentage of ovarian tumors. The IGF-II receptor acts as a sink for excess IGF-II, and, thus, its down-regulation, functional mutation, or genetically engineered loss can result in increased IGF-II availability. An increase in local IGF-II either through increased expression or decreased receptor uptake and degradation could increase signaling through the insulin receptor as well as the more commonly considered IGF-I receptor (48). This finding would have obvious implications for strategies designed to interrupt signaling through the IGF-I receptor that do not consider insulin receptor-mediated mitogenic signals initiated by IGF-II. Further integrated studies of insulin and IGF-II ligands and receptors in ovarian tumors will be necessary to establish their role in the biology of epithelial ovarian cancer and to provide possible targets for innovative therapeutic strategies.
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Acknowledgments |
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Footnotes |
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1 K.R.K. and O.I.F. contributed equally to this work. 
Abbreviations: FITC, Fluorescein isothiocyanate; OSE, ovarian surface epithelial; SFM, serum-free medium.
Received December 17, 2001.
Accepted for publication May 7, 2002.
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