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Specific binding sites for insulin and insulin-like growth factor I in human endometrial cancer
Specific binding sites for insulin and insulin-like growth factor I in human endometrial cancer Manubai Nagamani, MD, Charles A. Stuart, MD, Patricia A. Dunhardt, BA, and Mark G. Doherty, MD Galveston, Texas Insulin and insulin-like growth factor I are known to be mitogenic and therefore may playa role in the development of endometrial cancer. We undertook this study to investigate whether human endometrial cancer tissue has receptors for these substances. Endometrial cancer tissue samples were obtained at hysterectomy from 10 women with endometrial cancer, and control endometrial tissue was collected from normal cycling women undergoing hysterectomy for non endocrine problems. Binding studies with iodine 125-insulin and ['25 1]insulin-like growth factor I revealed the presence of specific binding sites for insulin and insulin-like growth factor I in both normal endometrium and endometrial cancer tissue. The percent binding of ['25 1]insulin in the endometrial cancer tissue (mean ± SE 2.4% ± 0.5%/100 flog protein) was not significantly different from that in normal endometrium (3.5% ± 1%/100 flog protein). On the contrary, the percent total binding of [,25]insulin-like growth factor I in the endometrial cancer (5.3% ± 1.5%/100 flog protein) was significantly (p < 0.04) higher than that observed in normal endometrium (2.1% ± 0.4%/100 flog protein). There was a significant positive correlation between the histologic grade of the tumor and the insulin-like growth factor I binding (r = 0.865, P < 0.02). The affinity constants for the high-affinity receptors were similar in the normal and neoplastic endometrium. These results indicate that insulin and insulin-like growth factor I may playa role in the growth and development of endometrial cancer. (AM J OBSTET GVNECOL 1991;165:1865-71.)
Key words: Insulin receptor, insulin-like growth factor I receptors, endometrial cancer Postmenopausal women with endometrial cancer have increased serum insulin levels, and their insulin response to glucose ingestion is increased. ' Insulin and insulin-like growth factors (IGF) (specificially IGF-I) have been shown to be mitogenic, and most types of tumor cells are dependent on insulin for their proliferation. 2. 4 Insulin and IGF-I receptors are closely related to tyrosine kinase family of oncogenes. 5 Insulin and IGF-I therefore may playa role in the growth and development of endometrial cancer. IGF-I messenger ribonucleic acid has been demonstrated in the rat uterus, and estrogens increase IGF-I messenger ribonucleic acid synthesis. 6 , 7 It is possible that IGF-I of uterine or extrauterine origin plays a role in the growth and proliferation of the endometrial cancer cells in an endocrine, autocrine, or paracrine fashion. We undertook this study to investigate (1) whether human endometrial cancer has binding sites for insulin From the Departments of Obstetrics and Gynecology and Medicine, The University of Texas Medical Branch. Supported by National Institutes of Health grants Nos. CA45181 and DK33749 and National Institutes of Health General Clinical Research Center grant No. M01-RR00073. Presented at the Thirty-eighth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 20-23, 1991. Reprint requests: Manubai Nagamani, MD, Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, TX 77550.
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and IGF-I, (2) if these sites are present, whether the concentrations of these receptors are increased in endometrial cancer tissue compared with normal endometrium, (3) whether the concentrations of these receptors correlate with the histologic grade of the tumor, and (4) whether there is an ip.crease in serum IGF-I levels in women with endometrial cancer.
Material and methods After the study was approved by the institutional review board, endometrial cancer tissue samples were obtained at hysterectomy from 10 patients with endometrial cancer. Normal endometrial tissue was obtained from women undergoing hysterectomy (in the proliferative phase of the cycle) for nonendocrine problems (cervical dysplasia, pelvic pain, pelvic relaxation). The tissue was washed in ice-cold buffer to remove any blood, quickly frozen in liquid nitrogen, and stored at - 72° C until use. The day before hysterectomy fasting blood samples were obtained from all patients with cancer for measurement of insulin, glucose, and IGF-I levels. Nine of the 10 patients with cancer were obese, weighing >20% above their ideal body weights. Ideal body weights were determined from the Metropolitan Life Insurance Company tables. The ages of the patients varied from 40 to 70 years. Two of the patients were premenopausal and had a history of oligomenorrhea since menarche. The rest of the cancer patients 1865
1866 Nagamani et al.
December 1991 Am J Obstet Gynecol
Table I. Clinical data for patients with endometrial cancer
% Ideal body weight
Fasting insulin* (pmollL)
Fasting IGF-It (ng/ml)
86 154 98 129 150 116 74 84 64 117
134 261 187 190 253 204 126 137 104 192
122 186 236 208 193 115 160 129 194 258
178 31 160 67 128 89 78 138 131 72
107 ± 10
179 ± 17
180 ± 16
107 ± 15
Age (yr)
Weight (kg)
J
59 62 51 40 57 57 45 51 70 54
Mean ± SE
55 ± 3
Patient
A B C D
E
F
G H I
*Normal range 36 to 107 pmol/L. tNormal range 36 to 233 ng/m!.
were 1 to 20 years postmenopausal and gave a history of regular menstrual cycles before the onset of menopause. Fasting insulin.levels were increased in all the patients, but fasting glucose levels were normal. Serum IGF-I levels were in the normal range. Clinical data on the endometrial cancer patients are presented in Table I. Binding studies. To prepare endometrial membranes, we used, with minor modification, a technique developed for cartilage plasma membranes." Endometrial tissue (400 to SOO mg) was homogenized in IS ml 0.01 moliL imidazole, 0.2S moliL sucrose, and O.OOS mol/L ethylenediaminetetraacetic acid, pH 7.S (ISE buffer), at 0° C for 60 seconds at half speed with a Polytron device (Brinkmann Instruments, Westbury, N.Y.). The homogenate was centrifuged at 1000g for IS minutes to remove nuclei, most mitochondria, and tissue fragments. The resulting supernatant was centrifuged at 3S,000g for 60 minutes to provide a final pellet that was resuspended in 0.001 mol/L imidazole, pH 7.S. Human insulin was the generous gift of Lilly Research Laboratories, Indianapolis. IGF-I and IGF-II were purchased from Bachem, Inc. (Torrance, Calif.). Insulin, IGF-I, and IGF-II were radiolabeled with iodine-12S with minor modifications of the dilute chloramine-T method of Freychet et al. 9 Insulin and IGFI binding studies were performed in 0.1 moliL N[2-hydroxyethyl]piperazine-N' -[2-ethant:sulfonic acid], 0.120 moliL sodium chloride, 0.0012 moliL magnesium sulfate, 0.001 moliL ethylenediametetraacetic acid; 0.01 mol/L glucose, O.OIS moliL sodium acetate, and 1% bovine serum albumin, pH S.O, as previously described. 1O Studies designed for Scatchard analysis of binding parameters consisted of eight different concentrations of insulin performed in triplicate. The concentrations of [125 I]insulin was approximately O.OS x 10- 9 mol/L, unlabeled porcine insulin was
added to achieve concentrations of 0,0.2, O.S, 1,2, 10, 20, 100, and 1000 x 10- 9 mol/L, and the membrane protein concentration was :;;1 mg/ml. For IGF-I binding studies unlabeled IGF-I and [l25I]IGF-I were used in similar concentrations. The mixtures were incubated at 4° C for IS to 18 hours, and the membranes were sedimented by centrifugation at IS,OOOg for IS minutes and washed once in binding buffer. The results are expressed as the percentage of total radioactivity bound per 100 ILg membrane protein. Protein was quantitated by the method of Lowry et aLl! Nonspecific binding was determined in the presence of at least 200fold molar excess of unlabeled insulin and IGF-I, respectively. Analysis of binding data was performed with a computer program designed to provide the best fit of a two-receptor model to the Scatchard plot. The program performs an iterative curve-stripping procedure in which a curvilinear Scatchard plot is stepwise recalculated, subtracting an increasing number of counts as a result of low-affinity specific binding until the optimal high-affinity straight line is achieved. lo Specificity studies. To evaluate the specificity of IGFI binding, endometrial membranes were incubated in the presence of a constant amount (1 x 105 cpm) of [125 I]IGF_I, with or without increasing concentrations of unlabeled IGF-II (1 to 100 nmoliL), IGF-I, or insulin (1 to 1000 nmoliL). To evaluate the specificity of insulin binding, the membranes were incubated in the presence of a constant amount (1 x 105 cpm) of [l 25 1]insulin with or without increasing concentrations of unlabeled insulin (1 to 1000 nmoliL, IGF-I (1 to 100 nmoliL), or IGF-II. Affinity labeling and gel electrophoresis. Specific labeling and cross-linking with disuccinimidyl suberate and electrophoresis were performed essentially as previously described for liver membranes,12 except separating gels were 5% polyacrylamide. Serum IGF-I levels were measured with a kit pur-
Receptors for insulin and IGF-I in endometrial cancer
Volume 165 Number 6, Part 1
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chased from Nichols Institute (San Juan Capistrano, Calif.) after extraction with acid ethanol to remove binding proteins. Insulin levels were measured by a previously described radioimmunoassay procedure." Independent Student t test was used to compare differences in specific binding between control endometrium and endometrial cancer. A p value of <0.05 was considered significant. Spearman's rank correlation was calculated between the histologic grade of the tumor and the specific binding of IGF-I and insulin to endometrial membranes. Results Insulin-binding studies. Specific binding sites for insulin were present in both the normal and cancerous endometrial tissue. The percent total binding of [125I]insulin m the endometrial cancer tissue (mean ± SE 2.4% ± 0.5%/100 fLg protein, range 0.9% to 9.0%1100 fLg protein) was not significantly different from that in the normal endometrium (mean ± SE 3.5% ± 1%1100 fLg protein, range 0.5% to 5.3%1100 fLg protein). Fig. 1 shows the competition curves for binding of [12 5I]insulin to endometrial membranes from normal endometrium and endometrial cancer tissue. The inset of Fig. 1 shows the mean data for Scatchard plots. The affinity constants for these high-affinity receptors were similar in the normal (4.0 ± 1.5 X 10" mol/L) and neoplastic (3.0 ± 0.6 x 109 mol/L) endometrium. The mean receptor concentration in the neoplastic endometrial tissue (0.11 ± 0.02 pmol/mg protein) was not significantly different from that in the normal endometrial tissue (0.33 ± 0.12 pmol/mg protein). There was no correlation between the stage or grade of the tumor and the insulin-binding activity. Specificity. Fig. 2 shows the competition curves for binding of [125 1]insulin to endometrial membranes pre-
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pared from normal and neoplastic endometrium in the absence and presence of varying concentrations of insulin, IGF-I, and IGF-II. Increasing concentrations of unlabeled insulin resulted in dose-dependent displacement of the specific insulin binding. IGF-II was approximately tenfold and IGF-II 100-fold less potent in competing for binding. Results of these specificity studies indicate that the endometrial membranes have specific receptors for insulin. IGF-I-binding studies. Specific binding sites for IGF-I were present in both normal and neoplastic endometrial tissue. Fig. 3 shows the competition curves
1868
Nagamani et al.
December 1991 Am J Obstet Gynecol
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for the binding of [125I]IGF_I to endometrial membranes from normal endometrium and endometrial cancer tissue. The inset of Fig. 3 shows the mean data for Scatchard plots. The percent of total binding of IGF-I III the normal endometrial tissue was 2.1 % ± 0.4% 1100 f-lg protein (range 0.5% to 4.2% 1100 f-lg protein). The percent of total binding of IGF-I in the endometrial cancer tissue (5.3% ± l.5%1100 I-l-g protein, range l.3% to 1l.9%1 100 I-l-g protein) was significantly higher (P < 0.04) than that in normal
endometrium. The affinity constants for these highaffinity receptors were similar (8.6 ± l.2 x 109 mol/L, normal endometrium; 8.1 ± l.8 x 10 9 mol/L, endometrial cancer tissue). The mean receptor concentration was 0.05 ± 0.03 pmol/mg membrane protein in the normal endometrium and 0.26 ± 0.1 pmol/mg protein in the neoplastic endometrium. There was a significant correlation between IGF-I binding in the endometrial cancer tissue and the histologic grade of the tumor (r = 0.864, P < 0.02) (Fig. 4). Specificity. The results of specificity studies on IGF-I binding are shown in Fig. 5. Increasing concentrations of unlabeled IGF-I resulted in dose-dependent displacement of specific IGF-I binding. IGF-II was fivefold less potent in its ability to compete for [125 I]IGF_I binding. Insulin was able to compete for binding at higher concentrations and produced 50% displacement at 300 nmol/L concentration. Autoradiography of dried polyacrylamide gels demonstrated specific labeling of bands of 130,000 and >300,000 molecular weight for radiolabeled insulin and IGF-I. Unlabeled insulin prevented 61 % of the labeling with [125 I]IGF-I, whereas unlabeled IGF-I prevented 88% of the labeling with [125 I]insulin (Fig. 6). The 130,000-molecular-weight band is believed to be the binding subunit of type I IGF receptor.' The specifically labeled larger-molecular-weight material probably represents subunits that are cross-linked together by disuccinimidyl suberate. Two bands of specific insulin binding with apparent molecular weights of 130,000 and >300,000 are similar to those observed for insulin receptors in rat liver and human placental membranes.,,12
Receptors for insulin and IGF-I in endometrial cancer
Volume 165 Number 6, Part 1
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Comment The association of obesity and endometrial cancer is well known. 14 Obesity is associated with insulin resistance and hyperinsulinemia. We have previously reported that insulin levels are increased in postmenopausal women with endometrial canceL 1 Premenopausal women with polycystic ovarian disease have hyperinsulinemia; these women are at increased risk for development of endometrial cancer at an earlier age. 15 The significance of this association of hyperinsulinemia and endometrial cancer, however, is not well understood. Prolonged administration of pharmacologic doses of insulin was found to exert a carcinogenic effect in mice. 16 Mammary carcinomas induced in rats were found to be dependent on insulin, and most of these tumors regressed in size or grew slower when the animals were deprived of insulin with administration of alloxan. 17 Insulin stimulates growth of numerous cell lines in vitro, including breast cancer cells. 18 Because the results of our study show that the neoplastic endometrial tissue contains specific high-affinity binding sites for insulin, insulin might playa role in the growth or development of endometrial cancer as well. Surrey et al. 19 observed that insulin and IGF-I stimulate proliferation of endometrial stromal cells in culture. There has been only one previous study on insulin binding in the human endometrium. Sheets et al. 20 reported the presence of specific binding sites for insulin in the normal human endometrial tissue. Our results in normal endometrium are similar to those reported in this pre-
45 Fig. 6. Autoradiograph of 3-(trimethylsilyl)-I-propane-sulfonic acid and cross-linked affinity labeling of insulin and IGFI receptors in endometrial membranes. Shown here is autoradiograph of endometrial membranes labeled with [125 IJinsulin and IGF-I in presence or absence of unlabeled ligands. Samples were reduced with mercaptoethanol and electrophoresed through polyacrylamide gels as described in Material and methods. Lane 1 a contains radiolabeled insulin that is cross-linked in absence of unlabeled ligand, whereas lane 1 b sample was bound in presence of 100 nmoll L unlabeled insulin and lane lc shows labeling in presence of 100 nmollL unlabeled IGF-I. Lane 2a shows labeling with [125 IJIGF-I in absence of unlabeled ligand. Lanes 2b and 2c show labeling with [12'IJIGF-I in presence of 100 nmollL unlabeled insulin or IGF-I, respectively. Specifically labeled large-molecularweight material probably represents subunits that are crosslinked together by disuccinimidyl suberate. There is no evidence of IGF-II receptor or specifically labeled binding proteins.
vious study. We have shown for the first time that human endometrial cancer has specific binding sites for insulin. The mean insulin receptor concentration in the endometrial cancer was slightly lower than that in the normal endometrium, but the difference was not statistically significant. The decrease in insulin receptor concentration could be due to down-regulation that is a secondary effect of the elevated peripheral insulin levels. Since only a small percentage of insulin receptors are occupied at insulin concentrations that have maximal biologic effects, a decrease in receptor binding would not be expected to change the maximal effect of insulin!1 Insulin in high concentrations binds to IGFI receptors!2 The effect of insulin therefore might be mediated either through its own receptor or by cross-
1870
Nagamani et al.
over stimulation through IGF-I receptor. Our specificity data do support possible crossover action. The other possible role of insulin in the pathogenesis of endometrial cancer is to increase the aromatization of androgens in the endometrial tissue. Tseng et aU 3 reported that the endometrium has the ability to convert androgens to estrogen by aromatization. Randolph et aI!4 observed that the aromatase activity in the human endometrial glands and stroma increased proportionately with increasing concentrations of insulin. 24 Hyperinsulinemia can therefore lead to an increase in estrogen production at the endometrial tissue level. The human endometrial tissue has specific high-affinity binding sites for IGF-I; therefore IGF-I of intrauterine or extrauterine origin might playa role in the growth and proliferation of endometrial cancer cells. Because the peripheral IGF-I levels were not significantly different between patients with cancer and controls, IGF-I produced locally in the endometrial tissue may be acting in an autocrine or paracrine fashion. Breast cancer cells have been shown to secrete IGF-I, and 17j3-estradiol stimulates IGF-I production by these cancer cells. 25 Endometrial cancer, like breast cancer, is an estrogen-dependent cancer. Studies by Murphy et al. 6. 7 indicate that estrogen induces IGF-I expression in the rat uterus. Estrogen-induced proliferation of endometrial cells might be mediated through IGF-I. The binding activity of IGF-I was significantly higher in the neoplastic endometrium than in normal endometrium. Lack of a difference in receptor affinity, as shown by the Scatchard plots, indicates that the increasing binding is primarily due to an increase in the number of receptors. The fact that there was a significant positive correlation between the histologic grade of the tumor and the IGF-I binding indicates that IGFI may playa role in the proliferation of the tumor cells. Undifferentiated tumors, which have increased miototic activity, had an increased number of binding sites for IGF-I when compared with the number in the welldifferentiated tumors. Surrey et al. 19 observed increased tritium-thymidine incorporation on addition of IGF-I to endometrial stromal cell cultures. The only previous report on IGF-I binding in endometrial cancer tissue is that of Talavera et al. 26 Our results are similar to this previous report, and these authors also observed increased IGF-I binding in undifferentiated tumors. Rutanen et aU7 looked at the IGF-I receptor and IGF-I-binding protein concentrations in the normal human endometrium at different phases of the menstrual cycle. These authors observed that there is an increase in the number of IGF-I receptors and 34K IGF-I-binding protein in the late secretory phase endometrium and that the binding protein competes with membrane receptors for IGF-I binding. There was no binding protein present in the proliferative phase endometrium. 27 In our study all the normal endometrial
December 1991 Am J Obstet Gynecol
tissue samples studied were in the proliferative phase of the cycle. Because 8 of the 10 cancer patients were postmenopausal and the two premenopausal patients had anovulatory cycles, there was probably no 34K IGF -binding protein present in the cancer tissue and all the IGF-I receptors were probably available for IGFI action. However, this needs to be confirmed by further studies on IGF-binding proteins in endometrial cancer tissue. In conclusion, results of our study indicate that endometrial cancer tissue has specific binding sites for both insulin and IGF-I. Because most of the women with endometrial cancer are obese and have hyperinsulinemia, insulin may playa role in the growth and development of this tumor. Since we did not find an increase in peripheral IGF-I levels in women with endometrial cancer, it is possible that IGF-I is produced locally in the tissue and acts in an autocrine or paracrine fashion.
REFERENCES 1. Nagamani M, Hannigan EV, Dinh TV, Stuart CA. Hyperinsulinemia and stromal luteinization of the ovaries in women with endometrial cancer. J Clin Endocrinol Metab 1988;67: 1,144-8. 2. Straus DS. Growth stimulating actions of insulin in vitro and in vivo. Endocrinol Rev 1984;5:356-69. 3. Osborne CK, Bolan G, Monaco ME, Lippman ME. Hormone responsive human breast cancer in long-term tissue culture: effect of insulin. Proc Nat! Acad Sci USA 1978;73:4536-40. 4. Furlanetto RW, Dicarlo IN. Somatomedin-C receptors and growth effects in human breast cells maintained in long term tissue culture. Cancer Res 1984;44:2122-8. 5. Ullrich A, BellJR, Chen EY, et al. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature 1985;313:756-61. 6. Murphy LJ, Murphy LC, Friesen HG. Estrogen induces insulin-like growth factor-I expression in the rat uterus. Mol Endocrinol 1987;1:445-50. 7. Murphy LJ, Friesen HG. Differential effects of estrogen and growth hormone on uterine and hepatic insulin-like growth factor-I gene expression in the ovariectomized hypophysectomized rat. Endocrinology 1988; 12 :32532. 8. Stuart CA, Furlanetto RW, Lebovitz HE. The insulin receptor for embryonic chick cartilage. Endocrinology 1979; 105: 1293-302. 9. Freychet P, Roth J, Neville DM. Monoidoinsulin: demonstration of its biologic activity and binding to fat cells and liver membranes. Biochem Biophys Res Commun 1971;43:400-8. 10. Stuart CA. Phylogenetic distance from man correlates with immunologic cross-reactivity among liver insulin receptors. Comp Biochem Physiol 1986;84B:167-72. 11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurements with the Folin phenol reagent. J Bioi Chern 1951; 193:267-75. 12. Stuart CA. Characteristics of a novel insulin receptor from stingray liver. J Bioi Chern 1988;263:7881-6. 13. Soeldner JS, Slone D. Critical variables in the radioimmunoassay of serum insulin using the double antibody technique. Diabetes 1965;14:771-9. 14. Wynder EL, Escher GC, Mantel N. An epidemiological investigation of cancer of the endometrium. Cancer 1966; 19:489-520.
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15. Jackson RL, Doherty MB. The Stein-Leventhal syndrome: analysis of 43 cases with special reference to association with endometrial carcinoma. AM ] OBSTET GYNECOL 1957;73:161-73. 16. Lupulescu AP. Effect of prolonged insulin treatment on carcinoma formation in mice. Cancer Res 1985;45:328895. 17. Hueson ]C, Legros N. Influence of insulin deprivation on growth of the 7,12-dimenthyl-benz(a)anthracene-induced mammary carcinoma in rats subjected to alloxan diabetes and food restriction. Cancer Res 1972;31:22632. 18. Osborne KC, Bolar G, Monaco ME, Lippman ME. Hormone responsive human breast cancer in long term tissue culture. Cell Bioi 1976;73:4536-40. 19. Surrey E, Rutanen EM, Halme J. Effects of insulin like growth factor-I (IGF-I) on endometrial stromal cell proliferation in vitro [Abstract 506]. In: Proceedings of the thirty-eighth annual meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 20-23, 1991. San Antonio: Society for Gynecologic Investigation, 1991 :334. 20. Sheets EE, Tsibris ]CM, Cook NI, Virgin DS, DeMay RM, Spellacy WN. In vitro binding of insulin and epidermal growth factor to human endometrium and endocervix. AM] OBSTET GYNECOL 1985;153:60-5.
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21. Kono T, Barbam FW. The relationship between the insulin binding capacity of fat cells and the cellular response to insulin.] Bioi Chern 1971;426:6210-6. 22. Rechler MM. The nature and regulation of the receptors for insulin-like growth factors. Annu Rev Physiol 1985;47:425-42. 23. Tseng L, Mazella .1, Mann W.1, Thomas J. Estrogen synthesis in normal and malignant human endometrium. .1 Clin Endocrinol Metab 1982;55:1029-31. 24. Randolph ]F, Kipersztok S, Ayers ]WT, Ansbacher R, Peegel H, Menon KM.1. The effect of insulin on aromatase activity is isolated human endometrial glands and stroma. AM] OBSTET GYNECOL 1987;157:1534-9. 25. Huff K, Knabbe C, Lindsey R, et al. Multihormonal regulation of insulin-like growth factor-I related protein in MCF-7 human breast cancer cells. Mol Endocrinol 1988;2:200-8. 26. Talavera F, Reynolds KR, Roberts ]A, Menon KMJ. Insulin-like growth factor-I receptors in normal and neoplastic human endometrium. Cancer Res 1990;50:301924. 27. Rutanen EM, Pekonen F, Makinen T. Soluble 34K binding protein inhibits the binding of insulin like growth factor I to its cell receptors in human secretory phase endometrium. ] Clin Endocrinol Metab 1988;66: 173-80.
Key words: Insulin receptor, insulin-like growth factor I receptors, endometrial cancer Postmenopausal women with endometrial cancer have increased serum insulin levels, and their insulin response to glucose ingestion is increased. ' Insulin and insulin-like growth factors (IGF) (specificially IGF-I) have been shown to be mitogenic, and most types of tumor cells are dependent on insulin for their proliferation. 2. 4 Insulin and IGF-I receptors are closely related to tyrosine kinase family of oncogenes. 5 Insulin and IGF-I therefore may playa role in the growth and development of endometrial cancer. IGF-I messenger ribonucleic acid has been demonstrated in the rat uterus, and estrogens increase IGF-I messenger ribonucleic acid synthesis. 6 , 7 It is possible that IGF-I of uterine or extrauterine origin plays a role in the growth and proliferation of the endometrial cancer cells in an endocrine, autocrine, or paracrine fashion. We undertook this study to investigate (1) whether human endometrial cancer has binding sites for insulin From the Departments of Obstetrics and Gynecology and Medicine, The University of Texas Medical Branch. Supported by National Institutes of Health grants Nos. CA45181 and DK33749 and National Institutes of Health General Clinical Research Center grant No. M01-RR00073. Presented at the Thirty-eighth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 20-23, 1991. Reprint requests: Manubai Nagamani, MD, Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, TX 77550.
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and IGF-I, (2) if these sites are present, whether the concentrations of these receptors are increased in endometrial cancer tissue compared with normal endometrium, (3) whether the concentrations of these receptors correlate with the histologic grade of the tumor, and (4) whether there is an ip.crease in serum IGF-I levels in women with endometrial cancer.
Material and methods After the study was approved by the institutional review board, endometrial cancer tissue samples were obtained at hysterectomy from 10 patients with endometrial cancer. Normal endometrial tissue was obtained from women undergoing hysterectomy (in the proliferative phase of the cycle) for nonendocrine problems (cervical dysplasia, pelvic pain, pelvic relaxation). The tissue was washed in ice-cold buffer to remove any blood, quickly frozen in liquid nitrogen, and stored at - 72° C until use. The day before hysterectomy fasting blood samples were obtained from all patients with cancer for measurement of insulin, glucose, and IGF-I levels. Nine of the 10 patients with cancer were obese, weighing >20% above their ideal body weights. Ideal body weights were determined from the Metropolitan Life Insurance Company tables. The ages of the patients varied from 40 to 70 years. Two of the patients were premenopausal and had a history of oligomenorrhea since menarche. The rest of the cancer patients 1865
1866 Nagamani et al.
December 1991 Am J Obstet Gynecol
Table I. Clinical data for patients with endometrial cancer
% Ideal body weight
Fasting insulin* (pmollL)
Fasting IGF-It (ng/ml)
86 154 98 129 150 116 74 84 64 117
134 261 187 190 253 204 126 137 104 192
122 186 236 208 193 115 160 129 194 258
178 31 160 67 128 89 78 138 131 72
107 ± 10
179 ± 17
180 ± 16
107 ± 15
Age (yr)
Weight (kg)
J
59 62 51 40 57 57 45 51 70 54
Mean ± SE
55 ± 3
Patient
A B C D
E
F
G H I
*Normal range 36 to 107 pmol/L. tNormal range 36 to 233 ng/m!.
were 1 to 20 years postmenopausal and gave a history of regular menstrual cycles before the onset of menopause. Fasting insulin.levels were increased in all the patients, but fasting glucose levels were normal. Serum IGF-I levels were in the normal range. Clinical data on the endometrial cancer patients are presented in Table I. Binding studies. To prepare endometrial membranes, we used, with minor modification, a technique developed for cartilage plasma membranes." Endometrial tissue (400 to SOO mg) was homogenized in IS ml 0.01 moliL imidazole, 0.2S moliL sucrose, and O.OOS mol/L ethylenediaminetetraacetic acid, pH 7.S (ISE buffer), at 0° C for 60 seconds at half speed with a Polytron device (Brinkmann Instruments, Westbury, N.Y.). The homogenate was centrifuged at 1000g for IS minutes to remove nuclei, most mitochondria, and tissue fragments. The resulting supernatant was centrifuged at 3S,000g for 60 minutes to provide a final pellet that was resuspended in 0.001 mol/L imidazole, pH 7.S. Human insulin was the generous gift of Lilly Research Laboratories, Indianapolis. IGF-I and IGF-II were purchased from Bachem, Inc. (Torrance, Calif.). Insulin, IGF-I, and IGF-II were radiolabeled with iodine-12S with minor modifications of the dilute chloramine-T method of Freychet et al. 9 Insulin and IGFI binding studies were performed in 0.1 moliL N[2-hydroxyethyl]piperazine-N' -[2-ethant:sulfonic acid], 0.120 moliL sodium chloride, 0.0012 moliL magnesium sulfate, 0.001 moliL ethylenediametetraacetic acid; 0.01 mol/L glucose, O.OIS moliL sodium acetate, and 1% bovine serum albumin, pH S.O, as previously described. 1O Studies designed for Scatchard analysis of binding parameters consisted of eight different concentrations of insulin performed in triplicate. The concentrations of [125 I]insulin was approximately O.OS x 10- 9 mol/L, unlabeled porcine insulin was
added to achieve concentrations of 0,0.2, O.S, 1,2, 10, 20, 100, and 1000 x 10- 9 mol/L, and the membrane protein concentration was :;;1 mg/ml. For IGF-I binding studies unlabeled IGF-I and [l25I]IGF-I were used in similar concentrations. The mixtures were incubated at 4° C for IS to 18 hours, and the membranes were sedimented by centrifugation at IS,OOOg for IS minutes and washed once in binding buffer. The results are expressed as the percentage of total radioactivity bound per 100 ILg membrane protein. Protein was quantitated by the method of Lowry et aLl! Nonspecific binding was determined in the presence of at least 200fold molar excess of unlabeled insulin and IGF-I, respectively. Analysis of binding data was performed with a computer program designed to provide the best fit of a two-receptor model to the Scatchard plot. The program performs an iterative curve-stripping procedure in which a curvilinear Scatchard plot is stepwise recalculated, subtracting an increasing number of counts as a result of low-affinity specific binding until the optimal high-affinity straight line is achieved. lo Specificity studies. To evaluate the specificity of IGFI binding, endometrial membranes were incubated in the presence of a constant amount (1 x 105 cpm) of [125 I]IGF_I, with or without increasing concentrations of unlabeled IGF-II (1 to 100 nmoliL), IGF-I, or insulin (1 to 1000 nmoliL). To evaluate the specificity of insulin binding, the membranes were incubated in the presence of a constant amount (1 x 105 cpm) of [l 25 1]insulin with or without increasing concentrations of unlabeled insulin (1 to 1000 nmoliL, IGF-I (1 to 100 nmoliL), or IGF-II. Affinity labeling and gel electrophoresis. Specific labeling and cross-linking with disuccinimidyl suberate and electrophoresis were performed essentially as previously described for liver membranes,12 except separating gels were 5% polyacrylamide. Serum IGF-I levels were measured with a kit pur-
Receptors for insulin and IGF-I in endometrial cancer
Volume 165 Number 6, Part 1
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chased from Nichols Institute (San Juan Capistrano, Calif.) after extraction with acid ethanol to remove binding proteins. Insulin levels were measured by a previously described radioimmunoassay procedure." Independent Student t test was used to compare differences in specific binding between control endometrium and endometrial cancer. A p value of <0.05 was considered significant. Spearman's rank correlation was calculated between the histologic grade of the tumor and the specific binding of IGF-I and insulin to endometrial membranes. Results Insulin-binding studies. Specific binding sites for insulin were present in both the normal and cancerous endometrial tissue. The percent total binding of [125I]insulin m the endometrial cancer tissue (mean ± SE 2.4% ± 0.5%/100 fLg protein, range 0.9% to 9.0%1100 fLg protein) was not significantly different from that in the normal endometrium (mean ± SE 3.5% ± 1%1100 fLg protein, range 0.5% to 5.3%1100 fLg protein). Fig. 1 shows the competition curves for binding of [12 5I]insulin to endometrial membranes from normal endometrium and endometrial cancer tissue. The inset of Fig. 1 shows the mean data for Scatchard plots. The affinity constants for these high-affinity receptors were similar in the normal (4.0 ± 1.5 X 10" mol/L) and neoplastic (3.0 ± 0.6 x 109 mol/L) endometrium. The mean receptor concentration in the neoplastic endometrial tissue (0.11 ± 0.02 pmol/mg protein) was not significantly different from that in the normal endometrial tissue (0.33 ± 0.12 pmol/mg protein). There was no correlation between the stage or grade of the tumor and the insulin-binding activity. Specificity. Fig. 2 shows the competition curves for binding of [125 1]insulin to endometrial membranes pre-
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pared from normal and neoplastic endometrium in the absence and presence of varying concentrations of insulin, IGF-I, and IGF-II. Increasing concentrations of unlabeled insulin resulted in dose-dependent displacement of the specific insulin binding. IGF-II was approximately tenfold and IGF-II 100-fold less potent in competing for binding. Results of these specificity studies indicate that the endometrial membranes have specific receptors for insulin. IGF-I-binding studies. Specific binding sites for IGF-I were present in both normal and neoplastic endometrial tissue. Fig. 3 shows the competition curves
1868
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for the binding of [125I]IGF_I to endometrial membranes from normal endometrium and endometrial cancer tissue. The inset of Fig. 3 shows the mean data for Scatchard plots. The percent of total binding of IGF-I III the normal endometrial tissue was 2.1 % ± 0.4% 1100 f-lg protein (range 0.5% to 4.2% 1100 f-lg protein). The percent of total binding of IGF-I in the endometrial cancer tissue (5.3% ± l.5%1100 I-l-g protein, range l.3% to 1l.9%1 100 I-l-g protein) was significantly higher (P < 0.04) than that in normal
endometrium. The affinity constants for these highaffinity receptors were similar (8.6 ± l.2 x 109 mol/L, normal endometrium; 8.1 ± l.8 x 10 9 mol/L, endometrial cancer tissue). The mean receptor concentration was 0.05 ± 0.03 pmol/mg membrane protein in the normal endometrium and 0.26 ± 0.1 pmol/mg protein in the neoplastic endometrium. There was a significant correlation between IGF-I binding in the endometrial cancer tissue and the histologic grade of the tumor (r = 0.864, P < 0.02) (Fig. 4). Specificity. The results of specificity studies on IGF-I binding are shown in Fig. 5. Increasing concentrations of unlabeled IGF-I resulted in dose-dependent displacement of specific IGF-I binding. IGF-II was fivefold less potent in its ability to compete for [125 I]IGF_I binding. Insulin was able to compete for binding at higher concentrations and produced 50% displacement at 300 nmol/L concentration. Autoradiography of dried polyacrylamide gels demonstrated specific labeling of bands of 130,000 and >300,000 molecular weight for radiolabeled insulin and IGF-I. Unlabeled insulin prevented 61 % of the labeling with [125 I]IGF-I, whereas unlabeled IGF-I prevented 88% of the labeling with [125 I]insulin (Fig. 6). The 130,000-molecular-weight band is believed to be the binding subunit of type I IGF receptor.' The specifically labeled larger-molecular-weight material probably represents subunits that are cross-linked together by disuccinimidyl suberate. Two bands of specific insulin binding with apparent molecular weights of 130,000 and >300,000 are similar to those observed for insulin receptors in rat liver and human placental membranes.,,12
Receptors for insulin and IGF-I in endometrial cancer
Volume 165 Number 6, Part 1
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Comment The association of obesity and endometrial cancer is well known. 14 Obesity is associated with insulin resistance and hyperinsulinemia. We have previously reported that insulin levels are increased in postmenopausal women with endometrial canceL 1 Premenopausal women with polycystic ovarian disease have hyperinsulinemia; these women are at increased risk for development of endometrial cancer at an earlier age. 15 The significance of this association of hyperinsulinemia and endometrial cancer, however, is not well understood. Prolonged administration of pharmacologic doses of insulin was found to exert a carcinogenic effect in mice. 16 Mammary carcinomas induced in rats were found to be dependent on insulin, and most of these tumors regressed in size or grew slower when the animals were deprived of insulin with administration of alloxan. 17 Insulin stimulates growth of numerous cell lines in vitro, including breast cancer cells. 18 Because the results of our study show that the neoplastic endometrial tissue contains specific high-affinity binding sites for insulin, insulin might playa role in the growth or development of endometrial cancer as well. Surrey et al. 19 observed that insulin and IGF-I stimulate proliferation of endometrial stromal cells in culture. There has been only one previous study on insulin binding in the human endometrium. Sheets et al. 20 reported the presence of specific binding sites for insulin in the normal human endometrial tissue. Our results in normal endometrium are similar to those reported in this pre-
45 Fig. 6. Autoradiograph of 3-(trimethylsilyl)-I-propane-sulfonic acid and cross-linked affinity labeling of insulin and IGFI receptors in endometrial membranes. Shown here is autoradiograph of endometrial membranes labeled with [125 IJinsulin and IGF-I in presence or absence of unlabeled ligands. Samples were reduced with mercaptoethanol and electrophoresed through polyacrylamide gels as described in Material and methods. Lane 1 a contains radiolabeled insulin that is cross-linked in absence of unlabeled ligand, whereas lane 1 b sample was bound in presence of 100 nmoll L unlabeled insulin and lane lc shows labeling in presence of 100 nmollL unlabeled IGF-I. Lane 2a shows labeling with [125 IJIGF-I in absence of unlabeled ligand. Lanes 2b and 2c show labeling with [12'IJIGF-I in presence of 100 nmollL unlabeled insulin or IGF-I, respectively. Specifically labeled large-molecularweight material probably represents subunits that are crosslinked together by disuccinimidyl suberate. There is no evidence of IGF-II receptor or specifically labeled binding proteins.
vious study. We have shown for the first time that human endometrial cancer has specific binding sites for insulin. The mean insulin receptor concentration in the endometrial cancer was slightly lower than that in the normal endometrium, but the difference was not statistically significant. The decrease in insulin receptor concentration could be due to down-regulation that is a secondary effect of the elevated peripheral insulin levels. Since only a small percentage of insulin receptors are occupied at insulin concentrations that have maximal biologic effects, a decrease in receptor binding would not be expected to change the maximal effect of insulin!1 Insulin in high concentrations binds to IGFI receptors!2 The effect of insulin therefore might be mediated either through its own receptor or by cross-
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Nagamani et al.
over stimulation through IGF-I receptor. Our specificity data do support possible crossover action. The other possible role of insulin in the pathogenesis of endometrial cancer is to increase the aromatization of androgens in the endometrial tissue. Tseng et aU 3 reported that the endometrium has the ability to convert androgens to estrogen by aromatization. Randolph et aI!4 observed that the aromatase activity in the human endometrial glands and stroma increased proportionately with increasing concentrations of insulin. 24 Hyperinsulinemia can therefore lead to an increase in estrogen production at the endometrial tissue level. The human endometrial tissue has specific high-affinity binding sites for IGF-I; therefore IGF-I of intrauterine or extrauterine origin might playa role in the growth and proliferation of endometrial cancer cells. Because the peripheral IGF-I levels were not significantly different between patients with cancer and controls, IGF-I produced locally in the endometrial tissue may be acting in an autocrine or paracrine fashion. Breast cancer cells have been shown to secrete IGF-I, and 17j3-estradiol stimulates IGF-I production by these cancer cells. 25 Endometrial cancer, like breast cancer, is an estrogen-dependent cancer. Studies by Murphy et al. 6. 7 indicate that estrogen induces IGF-I expression in the rat uterus. Estrogen-induced proliferation of endometrial cells might be mediated through IGF-I. The binding activity of IGF-I was significantly higher in the neoplastic endometrium than in normal endometrium. Lack of a difference in receptor affinity, as shown by the Scatchard plots, indicates that the increasing binding is primarily due to an increase in the number of receptors. The fact that there was a significant positive correlation between the histologic grade of the tumor and the IGF-I binding indicates that IGFI may playa role in the proliferation of the tumor cells. Undifferentiated tumors, which have increased miototic activity, had an increased number of binding sites for IGF-I when compared with the number in the welldifferentiated tumors. Surrey et al. 19 observed increased tritium-thymidine incorporation on addition of IGF-I to endometrial stromal cell cultures. The only previous report on IGF-I binding in endometrial cancer tissue is that of Talavera et al. 26 Our results are similar to this previous report, and these authors also observed increased IGF-I binding in undifferentiated tumors. Rutanen et aU7 looked at the IGF-I receptor and IGF-I-binding protein concentrations in the normal human endometrium at different phases of the menstrual cycle. These authors observed that there is an increase in the number of IGF-I receptors and 34K IGF-I-binding protein in the late secretory phase endometrium and that the binding protein competes with membrane receptors for IGF-I binding. There was no binding protein present in the proliferative phase endometrium. 27 In our study all the normal endometrial
December 1991 Am J Obstet Gynecol
tissue samples studied were in the proliferative phase of the cycle. Because 8 of the 10 cancer patients were postmenopausal and the two premenopausal patients had anovulatory cycles, there was probably no 34K IGF -binding protein present in the cancer tissue and all the IGF-I receptors were probably available for IGFI action. However, this needs to be confirmed by further studies on IGF-binding proteins in endometrial cancer tissue. In conclusion, results of our study indicate that endometrial cancer tissue has specific binding sites for both insulin and IGF-I. Because most of the women with endometrial cancer are obese and have hyperinsulinemia, insulin may playa role in the growth and development of this tumor. Since we did not find an increase in peripheral IGF-I levels in women with endometrial cancer, it is possible that IGF-I is produced locally in the tissue and acts in an autocrine or paracrine fashion.
REFERENCES 1. Nagamani M, Hannigan EV, Dinh TV, Stuart CA. Hyperinsulinemia and stromal luteinization of the ovaries in women with endometrial cancer. J Clin Endocrinol Metab 1988;67: 1,144-8. 2. Straus DS. Growth stimulating actions of insulin in vitro and in vivo. Endocrinol Rev 1984;5:356-69. 3. Osborne CK, Bolan G, Monaco ME, Lippman ME. Hormone responsive human breast cancer in long-term tissue culture: effect of insulin. Proc Nat! Acad Sci USA 1978;73:4536-40. 4. Furlanetto RW, Dicarlo IN. Somatomedin-C receptors and growth effects in human breast cells maintained in long term tissue culture. Cancer Res 1984;44:2122-8. 5. Ullrich A, BellJR, Chen EY, et al. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature 1985;313:756-61. 6. Murphy LJ, Murphy LC, Friesen HG. Estrogen induces insulin-like growth factor-I expression in the rat uterus. Mol Endocrinol 1987;1:445-50. 7. Murphy LJ, Friesen HG. Differential effects of estrogen and growth hormone on uterine and hepatic insulin-like growth factor-I gene expression in the ovariectomized hypophysectomized rat. Endocrinology 1988; 12 :32532. 8. Stuart CA, Furlanetto RW, Lebovitz HE. The insulin receptor for embryonic chick cartilage. Endocrinology 1979; 105: 1293-302. 9. Freychet P, Roth J, Neville DM. Monoidoinsulin: demonstration of its biologic activity and binding to fat cells and liver membranes. Biochem Biophys Res Commun 1971;43:400-8. 10. Stuart CA. Phylogenetic distance from man correlates with immunologic cross-reactivity among liver insulin receptors. Comp Biochem Physiol 1986;84B:167-72. 11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurements with the Folin phenol reagent. J Bioi Chern 1951; 193:267-75. 12. Stuart CA. Characteristics of a novel insulin receptor from stingray liver. J Bioi Chern 1988;263:7881-6. 13. Soeldner JS, Slone D. Critical variables in the radioimmunoassay of serum insulin using the double antibody technique. Diabetes 1965;14:771-9. 14. Wynder EL, Escher GC, Mantel N. An epidemiological investigation of cancer of the endometrium. Cancer 1966; 19:489-520.
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15. Jackson RL, Doherty MB. The Stein-Leventhal syndrome: analysis of 43 cases with special reference to association with endometrial carcinoma. AM ] OBSTET GYNECOL 1957;73:161-73. 16. Lupulescu AP. Effect of prolonged insulin treatment on carcinoma formation in mice. Cancer Res 1985;45:328895. 17. Hueson ]C, Legros N. Influence of insulin deprivation on growth of the 7,12-dimenthyl-benz(a)anthracene-induced mammary carcinoma in rats subjected to alloxan diabetes and food restriction. Cancer Res 1972;31:22632. 18. Osborne KC, Bolar G, Monaco ME, Lippman ME. Hormone responsive human breast cancer in long term tissue culture. Cell Bioi 1976;73:4536-40. 19. Surrey E, Rutanen EM, Halme J. Effects of insulin like growth factor-I (IGF-I) on endometrial stromal cell proliferation in vitro [Abstract 506]. In: Proceedings of the thirty-eighth annual meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 20-23, 1991. San Antonio: Society for Gynecologic Investigation, 1991 :334. 20. Sheets EE, Tsibris ]CM, Cook NI, Virgin DS, DeMay RM, Spellacy WN. In vitro binding of insulin and epidermal growth factor to human endometrium and endocervix. AM] OBSTET GYNECOL 1985;153:60-5.
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21. Kono T, Barbam FW. The relationship between the insulin binding capacity of fat cells and the cellular response to insulin.] Bioi Chern 1971;426:6210-6. 22. Rechler MM. The nature and regulation of the receptors for insulin-like growth factors. Annu Rev Physiol 1985;47:425-42. 23. Tseng L, Mazella .1, Mann W.1, Thomas J. Estrogen synthesis in normal and malignant human endometrium. .1 Clin Endocrinol Metab 1982;55:1029-31. 24. Randolph ]F, Kipersztok S, Ayers ]WT, Ansbacher R, Peegel H, Menon KM.1. The effect of insulin on aromatase activity is isolated human endometrial glands and stroma. AM] OBSTET GYNECOL 1987;157:1534-9. 25. Huff K, Knabbe C, Lindsey R, et al. Multihormonal regulation of insulin-like growth factor-I related protein in MCF-7 human breast cancer cells. Mol Endocrinol 1988;2:200-8. 26. Talavera F, Reynolds KR, Roberts ]A, Menon KMJ. Insulin-like growth factor-I receptors in normal and neoplastic human endometrium. Cancer Res 1990;50:301924. 27. Rutanen EM, Pekonen F, Makinen T. Soluble 34K binding protein inhibits the binding of insulin like growth factor I to its cell receptors in human secretory phase endometrium. ] Clin Endocrinol Metab 1988;66: 173-80.