[CANCER RESEARCH 53, 5585~5588, December l, 1993]
`
`Advances in Brief
`
`Insulin-like Growth Factor I Gene Expression in the Uterus Is Stimulated by
`
`Tamoxifen and Inhibited by the Pure Antiestrogen ICI 1827801
`
`Hung T. Huynh and Michael Pollalir2
`Departments of Medicine and Oncology, McGill University, and the Lady Davis Research Institute of the Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec,
`Canada HST 1E2
`
`Abstract
`
`Estrogen-induced uterine insulin-like growth factor I (IGF-I) expres-
`sion has been demonstrated to mediate at least in part the uterotrophic
`action of estradiol. We studied the effects of tamoxifen, a partial antago-
`nist to the estrogen receptor widely used in the treatment of breast cancer,
`and ICI 182780, a pure antagonist to the estrogen receptor, on uterine
`weight and uterine IGF-I gene expression in the rat. Tamoxifen increased
`uterine weight to 125% of control values and doubled uterine IGF-I
`expression. In contrast, ICI 182780 reduced uterine weight to 60% of
`control and uterine lGF-l gene expression to 13 % of control. These results
`demonstrate for the first time that uterine IGF-I expression is a molecular
`marker that correlates with the effects of partial agonists and antagonists
`to the estrogen receptor on the uterus. Furthermore, the induction of
`uterine IGF—I expression by tamoxifen provides a molecular mechanism to
`account for the uterotrophic effects which are commonly seen with
`tamoxifen therapy and which have been associated with endometrial neo-
`plasia.
`
`Introduction
`
`While the growth response of the uterus to estradiol has been
`recognized for many years, it is only recently that the role of IGF-I3
`as a mediator of estrogen-stimulated uterine proliferation has been
`appreciated (1, 2). Tamoxifen, a partial antagonist to the estrogen
`receptor, is widely used clinically because of its demonstrated efficacy
`in the adjuvant and palliative treatment of breast cancer and is cur-
`rently being evaluated in large-scale clinical trials as a breast cancer
`preventative agent (3). A uterotrophic effect of tamoxifen has been
`described in a variety of experimental systems (reviewed in Ref. 3),
`but effects of this compound and other anticstrogens on uterine IGF—I
`physiology have not been described. While tamoxifen therapy is gen-
`erally well
`tolerated, postmenopausal women receiving tamoxifen
`show estrogenic-like changes in the genital tract (4), and use of the
`drug is occasionally associated with endometrial neoplasia (5—7).
`Recognition of these issues has had a major impact on the design of
`tamoxifen prevention trials. The National Surgical Adjuvant Breast
`Project (NSABP) trial requires that participants undergo annual gy—
`necological examinations, while an Italian tamoxifen prevention trial
`is open only to women with a prior hysterectomy.
`We recently demonstrated that tamoxifen reduces growth hormone
`output by the pituitary gland (8, 9) and decreases hepatic IGF-I gene
`expression in experimental animals (10) and serum IGF-I concentra-
`tion in humans when used in both the adjuvant (11, 12) and preven—
`
`tative (13) settings. Furthermore, we have shown that the inhibitory
`effect of tamoxifen on hepatic IGF-I expression cannot be entirely
`accounted for by reduced growth hormone output (10), implying a
`pituitary-independent inhibitory effect of the drug on IGF-I expression
`in liver. In view of the clinical and animal model evidence for a
`
`uterotrophic effect of tamoxifen, we hypothesized that the effects of
`tamoxifen on IGF—I expression in the uterus are opposite from the
`inhibitory effects seen in liver. To investigate this possibility, we used
`an in viva rat model system to study the effects of tamoxifen and the
`pure antiestrogen ICI 182780 (14) on uterine weight and uterine lGF—I
`gene expression.
`
`Materials and Methods
`
`Animals, Drug Administration, and Sample Collection. Pituitary intact
`and hypox female Sprague-Dawley rats (50 days old; Charles River) were
`used. We studied control, tamoxifen-treated, and ICI 182780-treated intact
`animals and similarly treated hypophysectomized and hypophysectomized,
`growth hormone-replaced animals, for a total of 9 experimental groups. Each
`experimental group consisted of 4 animals. Tamoxifen and ICI 182780 were
`administered using a dose and route previously shown to have inhibitory ef-
`fect on 7,12—dimethylbcnz(a)anthracene-induced mammary tumors (15). Spe-
`cifically, 5 mg tamoxifen (Sigma Chemical Co., St. Louis, MO) or 5 mg ICI
`182780 (a generous gift from Dr. A. Wakeling, ICI Pharmaceuticals) were in-
`jected so. in 0.2 ml peanut oil once daily on 2 consecutive days. Control rats
`were given 5.0. 0.2 n11 peanut oil at the same time points. All the animals
`were sacrificed by C02 exposure 7 days after the first day of treatment. Hy-
`pophysectomized (Hypox) rats were used 2 weeks after hypophysectorny. For
`the growth hormone replacement experiments, rats were not only treated with
`tamoxifen (2 injections for a total of 10 mg on 2 consecutive days) or peanut
`oil as described above but also were given daily either i.p. saline or human
`recombinant growth hormone (kindly provided by Genentech, South San
`Francisco, CA), 100 jig/100 g body weight dissolved in saline i.p. for 7 days,
`starting on the same day as the tamoxifen or peanut oil. No other pituitary-
`dependent hormones were administered to hypox animals. All animals were
`sacrificed by carbon dioxide exposure on day 8. The uteri were excised, im-
`mediately frozen in liquid nitrogen, weighed and stored at —70°C for subse-
`quent RNA extraction. Animal experimental protocols were approved by the
`local animal care committee.
`
`Received 8/18/93; accepted 10/19/93.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`1 Supported by a grant from the National Cancer Institute of Canada to M. P. and by
`the Reisman Family Foundation.
`2To whom request for reprints should be addressed, at Lady Davis Institute for
`Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote Ste-
`Catherine Road, Montreal, Quebec, Canada H3T 1E2.
`3 The abbreviations used are: lGF-l, insulin-er growth factor I; TAM, tamoxifen;
`hypox, hypophyscctomizcd; cDNA, complementary DNA; SDS, sodium dodecyl sulfate;
`SSC, saline-sodium citrate; ADU, arbitrary density units; GH, growth hormone treated;
`ICI, ICI 182780.
`
`RNA Extraction and Hybridization. Total RNA was isolated from uteri
`using RNAZol premix solution and RNAZol B method (Tel-Test, Friend—
`swood, TX). For Northern blots, 60 ug of total uterine RNA were used per
`lane. Separate Northern blots were performed using RNA from each experi-
`mental animal. RNA was subjected to electrophoresis through 1.2% agarose
`gels containing 2.2% formaldehyde. The RNA was transferred onto Zeta-probe
`membrane (Bio-Rad) in 50 mM NaOH. The blots were hybridized overnight
`with nick—translated 32P-labeled rat prepro-IGF—l cDNA (16) (kindly provided
`by Dr. L. Murphy), dextran sulfate (10%), 1% SDS, herring sperm DNA (500
`pig/ml), 0.9 M NaCl, 50 mM NaZHPO4-7H20, and 5 ITIM EDTA. The membranes
`were subjected to three washes at 42°C for 15 min each in solution A (2X
`SSC-0.1% SDS), solution B (0.5x SSC—O.1% SDS), solution C (0.1x SSC-
`0.1% SDS), respectively. A final wash was done at 60"C in solution C. The
`blots were air-dried and subjected to autoradiography for 1 to 3 days with
`intensifying screen at —80°C. To control for equal loading of wells, we com-
`pared total amounts of RNA present in different lanes by rehybridizing the
`blots with labeled B—actin cDNA (17). Quantitative analysis of gene expression
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`TAM.
`
`ICI 182780, AND UTERINE lGFel GENE EXPRESSION
`
`was accomplished by scanning autoradiograms densitometrically. For each
`lane, the Sum of the density of bands corresponding to transcripts hybridizing
`with the lGF-l probe was calculated, and this figure was adjusted for minor
`differences in RNA loaded (determined as noted above). For each experimen-
`tal group, we present results by showing a representative Northern blot from
`one of the four replicates and also show mean densitometry data from the
`replicates.
`
`Results
`
`The effects of tamoxifen and ICI 182780 on uterine weight in intact
`rats are illustrated in Fig. 1A (CI). While tamoxifen increased weight
`to approximately 125% of control, ICI 182780 had the opposite effect,
`with reduction of weight to approximately 60% of control values.
`Representative Northern blots (Fig. 2A) and the pooled densitometric
`quantification of IGF-I expression from individual Northern blots
`from each experimental animal (Fig. 1A, I) demonstrate the effect of
`the treatments on uterine IGF-I expression. Following ICI 182780
`treatment, uterine IGF—I mRNA levels decreased dramatically com-
`pared to control intact rats (Fig. 2A). While ICI 182,780 caused a
`decrease in IGF-I transcripts, TAM significantly enhanced IGF-I
`mRNA accumulation (Fig. 24). Three predominant transcripts with
`molecular weights of 7.5, 1.4, and 1.0 kilobases were observed. The
`predominant IGF—I transcripts affected following TAM treatment were
`those of low molecular weight ranging from 800 to 450 base pairs
`(Fig. 2A). Densitometric quantitation (the sum of the density of all
`bands) revealed that tamoxifen increased IGF—I expression from 31.7
`t 3.1 (SD) to 60 i 7.5 ADU, while ICI 182780 suppressed the
`expression of this gene to 4.2 i 1.5 ADU, representing 13% of
`control, a value even less than that seen in a separate ovariectomized
`pituitary intact control group, where relative expression was densito-
`metrically quantified as 7.0 i 3.8 ADU.
`Figs. 13 and 23 show the results of similar experiments to examine
`the effects of tamoxifen or ICI 182780 on uterine weight (Fig. 18, El),
`densitometric quantitation of uterine IGF-l transcripts (Fig. 13, I),
`and representative Northern blots (Fig. 2B) of IGF-I gene expression
`in hypox and hypox, growth honnone-replaced animals. As expected,
`hypox rats had reduced uterine weight and lGF-I expression compared
`to controls. Growth hormone treatment of hypox animals resulted in
`a small increase in uterine weight (from 184 i 19 to 216 t 26 mg)
`and an increase in relative IGF—I expression from 5.4 t 0.6 to 11.1 i
`1.2 ADU, considerably less than the expression of 31.7 observed in
`intact animals. Obviously, the growth hormone-replaced hypox ani—
`
`mals remained estrogen deficient, and this result is therefore consis-
`tent with the stimulatory role of estrogens in regulating uterine IGF-I
`expression as previously reported by Murphy and Ghahary (2).
`The stimulatory effects of tamoxifen on IGF-I expression and to a
`lesser extent on uterine weight seen in intact animals were also evident
`in the hypox and hypox-growth hormone groups. Tire percentages of
`increase in uterine weight achieved by tamoxifen were similar in
`intact (125%) and hypox (131%) animals. In hypox, growth hormone
`replaced animals, tamoxifen had a stronger stimulatory effect on IGF-I
`gene expression than in hypox animals without growth hormone re—
`placement, but this was not associated with a significant increase in
`tamoxifen-stimulated uterine weight gain. ICI 182780 reduced uterine
`weight of hypox animals, despite the absence of further reduction of
`IGF—I gene expression to below hypox levels. In the ICI 182780-
`treated group of hypox animals, growth hormone replacement was
`effective at
`increasing IGF-I gene expression, but the stimulatory
`effect of growth hormone replacement on weight was attenuated com-
`pared to vehicle-treated hypox, growth hormone-replaced animals.
`to
`The differences between the following groups with respect
`IGF—I gene expression were significant (P < 0.05, Mann-Whitney U
`test): control versus tam; control versus ICI; TAM versus ICI; con—
`trol versus hypox; hypox versus hypox-TAM; hypox versus hypox-
`GH; hypox-GH versus hypox-GH-TAM; hypox ICI versus hypox-
`GH—ICI; hypox-TAM versus hypox-GH-TAM.
`Tamoxifen significantly reduced the serum IGF-I level, as de—
`scribed in our previous report (10). With respect to total body weight,
`we observed the following. For intact rats: control, 218.6 i 15.3 g;
`TAM, 191.4 1 11.6 g; ICI, 212.6 i 14.4 g. For hypox rats: hypox,
`144 i 11 g; hypox-GH, 179.8 i 15.3 g; hypox-GH—TAM, 170.28 :
`12.6 g; hypox-TAM, 131.5 : 11.7 g; hypox-ICI, 156 i 18 g; hypox-
`GH-ICI, 187.6 i 15.3 g.
`Fig. 3 shows the relationship between uterine weight and uterine
`IGF—I gene expression in all experimental groups. The correlation
`(Spearman R) between weight and IGF-1 expression was 0.71
`(P < 0.01).
`
`Discussion
`
`Estradiol is known to achieve its uterotrophic effect at least in part
`by stimulating uterine IGF-I expression (2). Our data provide the first
`evidence to support the hypothesis that the uterotrophic effect of
`tamoxifen also involves IGF—l as a mediator. The precise mechanism
`
`500
`
`400
`
`300
`
`200
`
`100
`
`Fig. 1. (A) Effect of tamoxifen and ICI 182780
`on uterine weight and uterine IGF—I expression in
`intact female rats. (B) Effect of tamoxifen and ICI
`182780 on uterine weight and uterine IGF—I expres-
`sion in hypox and hypox, growth hormone-replaced
`female rats. Treatments were administered as de-
`scribed in “Materials and Methods.” Gene expres-
`sion was quantified by densitornetric scanning of
`individual Northern blots obtained from individual
`animals in each experimental group. Mean values
`3 SD (bars) are plotted; each experimental group
`comprised 4 animals. C, control.
`
`
`
`Uterineweight(m3)
`
`70
`
`60
`
`50
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`40
`
`30
`
`20
`
`10
`
`
`
`uoisseidxeI'dSI
`
`i
`
`TAM
`
`C
`
`TAM
`
`iCi
`
`GH
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`GH-TAM GH-iCl
`
`D Uterine Weight I IGF-l expression
`
`Hypophysectomized
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`TAM.
`
`ICI 182780, AND UTERINE IGF-I GENE EXPRESSION
`
`
`
`
`
`(A) Representative Northern blots of
`Fig. 2.
`IGF—I mRNA and control ,B-actin mRNA [2.0 kilo-
`bases (kb)] expression in intact rats treated with
`vehicle (Lane I ), ICI 182780 (Lane 2), or tamoxi-
`fen (Lane 3). (3) Representative Northern blots of
`[GF—I mRNA and control ,B-actin mRNA (2.0 kb)
`expression in hypox and hypox, growth hormone-
`replaced rats. Lanes 1—3 are from hypox animals
`that received saline injections, while Lanes 4—6 are
`from hypox animals that received growth hormone
`replacement as described in “Materials and Meth-
`ods.” Lanes 1 and 4 are from vehicle—treated ani-
`mals, Lanes 2 and 5 are from ICI 182780—treated
`animals, and Lanes 3 and 6 are from tamoxifen-
`treated animals, as described in “Mater-iais and
`Methods."
`
`2.0:
`
`’
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`'
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`
`
`tagonist ICI 182780 is associated with inhibition of uterine IGF—I gene
`expression. The experimental manipulations involving various com-
`binations of growth hormone, tamoxifen, iCI 182780, and hypophy-
`sectomy resulted in a greater than 10-fold variation in uterine IGF-I
`expression. The correlation between uterine weight and IGF-1 expres-
`sion supports the hypothesis that the local expression of this gene is an
`important determinant of uterine growth. While uterine weight was in
`general well correlated with uterine IGF—I gene expression, it is note-
`worthy that in hypox and hypox-growth hormone-replaced animals,
`ICI 182780 administration suppressed uterine weight significantly,
`even though there was no significant effect of the drug on IGF—I
`expression in these animals, which had been rendered estrogen defi-
`cient by pituitary ablation. This suggests that ICI 182780 may, in
`addition to inhibiting IGF—I gene expression in uterus, alter the ex-
`pression of other genes involved in regulation of proliferation. We
`therefore are investigating the efiects of this compound on IGF-I
`receptor expression, IGF—I binding protein expression, and growth
`factors other than IGF-I.
`
`by which tamoxifen increases IGF-l gene expression is not well un-
`derstood, but IGF-I expression induced by tamoxifen may contribute
`to the estrogen-like effects of the drug. There is a precedent for this
`interpretation as Ignar-Trowbridge et at. (18) have demonstrated that
`EGF elicits estrogen—like action in the mouse uterus. The molecular
`basis for the opposite effects of tamoxifen on IGF-I expression in the
`uterus as compared to other tissues is under investigation and may be
`relevant to understanding the tissue-specific differences in agonist
`versus antagonist effects of the drug.
`As both estrogens and antiestrogens have important actions on the
`pituitary gland (19, 20), we considered the possibility that the effects
`of tamoxifen and/or ICI 182780 on the uterus were pituitary depen-
`dent. The results of our experiments with hypophysectomized animals
`suggest that growth hormone has a modest stimulatory effect on
`uterine IGF—I expression but that the effects of tamoxifen and ICI
`182780 on the uterus are not mediated via the pituitary. However, the
`data do demonstrate synergy between tamoxifen and growth hormone
`with respect to stimulation of uterine IGF-I gene expression, as by-
`pophysectomized animals treated with both growth hormone replace-
`ment and tamoxifen showed a level of uterine IGF-l expression
`greater than the sum of that observed in similar animals receiving
`growth hormone or tamoxifen individually. This observation is dis-
`tinct from that observed by Murphy et at. in an analogous experiment
`with estradiol (2) and suggests that the mechanism by which tamoxi-
`fen regulates uterine lGF—I expression may not be identical to that of
`estradio].
`We also demonstrate here for the first time that the reduction of
`
`While there is species-to-species variability in the tissue-specific
`predominance of agonist versus antagonist actions of tamoxifen at
`the estrogen receptor, the results reported here are relevant clinically,
`because women treated with tamoxifen frequently exhibit uterine hy-
`perplasia (4) and rarely show neoplasia (5—7). Tamoxifen has been
`proposed as a treatment for neoplastic conditions of the uterus (21),
`but results of clinical trials have not been impressive, and there are
`clinical and laboratory data suggesting that stimulation of cndome-
`trial neoplastic growth and leiomyoma growth by tamoxifen are pos-
`sible (22, 23). It is possible that the effect of tamoxifen on uterine
`uterine weight following treatment with the complete estrogen an—
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`

`TAM,
`
`ICI 182780, AND UTERINE lGF-I GENE EXPRESSION
`
`Inc._. for recombinant human growth hormone, and Dr. Richard
`Genentech,
`Margolese for useful discussion of the work.
`
`References
`
`o HYPOX-GH-TAM
`
`units
`UterinelGF-lgeneexpression,arbitarydensity
`
`
`
`
`
`HYPOXGH-ICI
`I
`
`HYP
`
`-
`9X ICI
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`O HVPOX-TAM
`. HYPOX-6H
`
`. HYPOX
`101
`
`. Murphy, L. J., Murphy, L. C., and Friesen, H. G. Estrogen induces insulinalike growth
`factorl expression in the rat uterus. Mol. Endocrinol., 1,: 445—450, 1987.
`Murphy, L., and Ghahary, A. Uterine insulin—like growth factor-1: regulation of
`expression and its role in estrogen-induced uterine proliferation. Endocr. Rev, 11:
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`Lemer, L., and Jordan, V. Development of antiestrogens and their use in breast cancer:
`Eighth Cain Memorial Award Lecture. Cancer Res, 50: 4177-4189, 1990.
`Fomander, T., Rutqvist, L. E., and Wilking, N. Effects of tamoxifen on the female
`genital tract. Ann. NYAcad. Sci., 622: 469—476, 1991.
`. Andersson, M., Storm, H. H., and Mouridser. H. T. Incidence of new primary cancers
`after adjuvant tamoxifen therapy and radiotherapy for early breast cancer. J. Natl.
`Cancer Inst., 83: 1013—1017, 1991.
`Fomander, T., Cedermark, B., Mattsson, S., Skoog, L, Theve, T., Askergren, J.,
`Rutqvist, L. E., Glas, U., Silfversward, C., Somell, A., Wilking, N., and Hjalmar, M.
`L. Adjuvant tamoxifen in early breast cancer: occurrence of new primary cancers.
`Lancet, 2: 117—120, 1989.
`Magriples, U., Naftolin, F., Schwartz, P. E., and Carcangiu, M. L. High-grade endo~
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`Tanneubaum, G. 5.. Gurd, W., Iapointe, M., and Pollak, M. Tamoxifen attenuates
`pulsatile growth hormone secretion: mediation in part by somatostatin. Endocrinol-
`ogy, 130: 3395—3401, 1992.
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`Huynh, H. T., Tetenes, E., Wallace, L., and Pollak, M. In vivo inhibition of insulin-like
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`Pollak, M. N., Custantino, J ., Polychronnkos, C., Blauer, S., Guyda, H., Redmond, (3.,
`Fisher, B., and Margolese, R. Effect of tamoxifen on serum insulin-like growth factor
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`Friedel, A., Jordan, V., and Pollak, M. Reduction in serum lGF—l levels following
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`l., Duckworth, M. L., and Friesen, H. 0. Identification,
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`
`0
`
`100
`
`200
`
`300
`
`400
`
`500
`
`Uterine weight, mg
`
`Fig. 3. Scatterplot relating uterine weight and uterine lGF—l gene expression for each
`experimental condition studied. The abbreviations used in identifying the points are: ICI,
`ICI-treated; TAM, tamoxifen treated, HYPOX, hypox; GH, growth hormone replaced;
`CNTRL, control.
`
`IGF-I gene expression which we describe here is related to these ad-
`verse effects of the drug. Because IGF-I responsivity has been re-
`ported for endometrial epithelial cells as well as uterine stromal and
`myometrial cells (2), our results suggest that inhibitory effects of ICI
`182780 on IGF—I gene expression may make this agent considerably
`more useful
`than tamoxifen in conditions such as endometriosis,
`leiomyomata, and endometrial carcinoma.
`Our data support the View that the use of complete estrogen an-
`tagonists instead of tamoxifen would eliminate adverse effects of
`anticstrogen therapy on the uterus. However, this approach might be
`problematic because it is likely that the beneficial effects of tamoxifen
`on serum lipids and bone density (reviewed in Ref. 3) would be lost.
`We have previously proposed that the coadministration of tamoxifen
`and a growth hormone—suppressive agent such as somatostatin or a
`growth hormone-releasing factor antagonist might improve the effi-
`cacy of tamoxifen by maximizing the reduction of extrauterine IGF-I
`expression associated with tamoxifen administration (11, 20). In view
`of the synergism between growth hormone and tamoxifen in stimu-
`lating uterine IGF-I gene expression reported here, it is possible that
`such a combination would also minimize the adverse effects of
`tamoxifen on the uterus.
`
`10.
`
`11.
`
`12‘
`
`13~
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`14,
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`15‘
`16.
`
`17
`
`18
`
`.
`
`'
`
`19
`
`.
`20~
`
`21.
`
`Acknowledgments
`
`22
`
`We thank Dr. L. Murphy for the rat lGF-I cDNA, Dr. Alan Wakeling for
`ICI 182780 and helpful comments, Laurie Wallace for technical assistance.
`
`23.
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`Cancer Research
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`AR
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`Insulin-like Growth Factor | Gene Expression in the Uterus ls
`Stimulated by Tamoxifen and Inhibited by the Pure
`Antiestrogen ICI 182780
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`Hung T. Huynh and Michael Pollak
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`Cancer Res 1993;53:5585-5588.
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