Growth Inhibition and Increase of Insulin Receptors in Antiestrogen-Resistant T47dcohuman Breast Cancer Cells by Progestins: Implications for Endocrine Therapies1

[CANCER RESEARCH 45,167-173, January 1985]

Growth Inhibition and Increase of Insulin Receptors in Antiestrogen-resistant T47DCOHuman Breast Cancer Cells by Progestins: Implications for Endocrine Therapies1

Kathryn B. Horwitz2 and Gary R. Freidenberg3

Departments oÃMedicine and Biochemistry, Biophysics, and Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262

ABSTRACT and the duration of remissions ranges from several months to 1 year or more (1, 25, 31, 33, 39, 43, 45). These results are There is renewed interest in the use of progestins to treat comparable to those obtained with antiestrogens, the most advanced breast cancer because results with these agents are common drug used for endocrine therapy (16, 39, 44). It is not comparable to those obtained with antiestrogens. However, it is known whether progestins act directly on the regressing tumors not known whether progestins inhibit the growth of breast tumor or indirectly through other hormones, (b) In support of the cells directly and independently of estradici. To study this, we hypothesis of Horwitz et al. (20), presence of PR4 may be the have used T47Dco human breast cancer cells. The progesterone single best marker for predicting both the hormone dependence receptors in these cells do not require estrogen induction, and of tumors and the disease-free survival of patients (11, 35). This this permits study of pure progestin effects without interference has led to speculation that PR-rich tumors would be especially by estradiol. We report here that, in the absence of estradici, sensitive to progestin treatment (26). (c) In small series of studies, physiological concentrations of progestins directly inhibit prolif positive responses to progestins have been obtained in tumors eration of these cells. At the same time, progestins increase the that have failed to respond to or have become resistant to levels of the receptors for insulin, a common cell mitogen. Ten antiestrogens or other endocrine therapies (14) or in tumors days of treatment with 1 or 10 nw of the synthetic progestin lacking ER (25, 39, 48). This suggests that some of the mecha R5020 suppresses cell growth approximately 50 to 60%. This is nisms responsible for the antitumor effects of progestins and consistent with the concentrations that either partially (approxi antiestrogens must differ. mately 10%) or more extensively (>60%) translocate cytoplasmic There is little experimental evidence underlying any of these progesterone receptors. Even a brief 1-hr pulse of R5020 has assumptions. It has been difficult to study the direct biological long-term growth-inhibitory effects. Progesterone is also antipro- actions of progestins separate from those of estrogens, because liferative, but its effects are attenuated because, unlike R5020, estrogen priming is required to maintain elevated, and presum it is rapidly metabolized in the medium. Other synthetic proges ably functional, levels of PR (19). Thus, the contaminating pres tins also inhibit cell growth, but unrelated steroids (estradiol, ence of estradiol in the system under study has precluded androgens, glucocorticoids, 1,25-dihydroxyvitamin D3) are inef accurate assessment of true progestin effects in vivo. Further fective. While growth is suppressed by R5020, insulin receptors more, for reasons that are not entirely clear, progesterone action increase rapidly and then fall to a new, elevated steady state as has also been difficult to study in vitro, where conditions can the cells slowly begin to proliferate. Only progestins have this usually be more carefully controlled. As a result, there are several effect on insulin receptors. We conclude that the hormonal established cell lines that are used for studies of glucocorticoid regulation of breast tumor cell growth is complex and includes and estrogen action in vitro and that contain estrogen-inducible progestins among the regulating factors. Furthermore, since PR but in which direct responses to progesterone have not been T47Dco cells are antiestrogen-resistant and estrogen receptor- demonstrable. negative, the antiproliferative effects of progestins must be me T47D is a relatively new human breast cancer cell line that has diated through mechanisms that differ from the cytotoxic effects PR and in which progesterone responses have recently been of antiestrogens. We propose that, clinically, antiestrogens and described (9, 10, 27, 49). In the "clone 11" subline, which has progestins may have complementary uses in breast cancer ER and responds to estrogens, progestins have antiestrogenic treatment, and we outline two therapeutic strategies. actions. They suppress growth-stimulatory effects of estrogens but have no growth effects alone (9,10). A variant subline in our INTRODUCTION laboratory, T47Dco, contains unusually high levels of PR that are independent of estrogen controls. The cells lack ER and are For several reasons, there is renewed interest in the use of antiestrogen resistant; their PR, although structurally and bio synthetic progestins to treat men and women who have ad chemically normal, are neither induced by estradiol nor sup vanced breast cancer, (a) Clinical results using pharmacological pressed by antiestrogens (17, 21, 22, 37). These cells can progestin concentrations have been very encouraging: the drugs therefore be used in estrogen-free conditions, to assess the are well tolerated, remissions occur in 35 to 45% of patients, direct biological actions of progestins, distinct from their anties trogenic ones. We have now studied the role of progestins on 1This work was supported by Grant CA-26869 from the NIH. 1Recipient of Research Career Development Award CA-00694 from the NIH. cell growth and on the receptors for insulin, a common cell To whom requests for reprints should be addressed. mitogen. The studies show that progestins alone inhibit cell 3Presentaddress: Departmentof Medicine,Universityof Californiaat San Diego, 4The abbreviations used are: PR, progesterone receptors; ER, estrogen recep La Jolla, CA 92093. ReceivedApril 2, 1984; accepted September 27, 1984. tors.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1985 American Association for Cancer Research. GROWTH REGULATION BY PROGESTINS growth and increase insulin receptor levels at concentrations RESULTS that bind to and translocate PR. These direct actions of proges tins, distinct from their antiestrogenic ones, have important im Progestins and Cell Growth. Chart 1 shows that the doubling plications for the design of endocrine therapies against breast time of log-phase T47DÂ«,cells in medium containing 5% char cancer, and 2 treatment strategies using progestins are de coal-stripped fetal calf serum is approximately 54 hr. Addition of scribed. 1 nw R5020, a synthetic progestin, slowed the doubling time to approximately 72 hr; at 10 nw R5020, the doubling time was 5 days. As a result, after 10 days, progestin-treated flasks had MATERIALS AND METHODS only 49% (1 nM R5020) and 39% (10 nw R5020) of the cell Hormones. [3H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20- number present in control flasks. Though cell growth is slowed dione) and unlabeted R5020 were obtained from New England Nuclear by the presence of R5020, the cells are not killed, but continue (Boston, MA). Other steroids were from Sigma Chemical Co. (St. Louis, to proliferate sluggishly through many passage generations. MO), except 1,25-dihydroxyvitamin D3, which was a gift from David Normal growth rate is gradually restored in such cells once the Feldman (Stanford University School of Medicine). 125l-Ai4porcine insulin hormone is removed (data not shown). (340 to 360 /iCi///g) and unlabeled insulin were supplied by Eli Lily Table 1 compares the growth-inhibitory properties of R5020 (Indianapolis, IN). with those of progesterone and shows, at the same concentra Cell Culture and Growth Studies. The routine culture of the T47D subline (T47DÅ“) of human breast cancer cells has been described (21). tions, the ability of the 2 hormones to translocate PR. The data Briefly, cells were grown in buffered RPMI Medium 1640 containing 5% show that, like R5020, progesterone also suppresses cell growth heat-inactivated fetal calf serum, nonessential amino acids, glutamine, but that it does so less efficiently. There are 2 reasons for this: penicillin, streptomycin, and porcine insulin (6 ng/ml). Stock cells were first, a higher concentration of progesterone than of R5020 is plated at a density of 4 x 10* celts (a 1:4 split) in 175-sq cm plastic flasks required to translocate the same number of cytoplasmic PR in and grown at 37Â°in a humidified atmosphere of 5% CO2 and air. For intact cells (Table 1); and, second, progesterone is rapidly (fw, growth studies, cells were harvested at confluence, replated in 25-sq cm approximately 2 hr) metabolized in the medium (22). In practice, flasks at the densities described in the figures for 18 to 48 hr, and then therefore, the cells are exposed to progesterone for only a few switched to modified insulin-free medium containing 5% heat-inactivated fetal calf serum stripped of endogenous hormones by 30 min of incuba tion at 45Â° with a dextran-coated charcoal pellet [0.25% Norit A, 0.0025% dextran in 0.01 M Tris-HCI (pH 8.0) at 4Â°;1 ml charcoal per 2 350 ml serum]. Steroids were prepared in ethanol at 1000-fold final concen tration and added to the medium. At harvest, the cells were suspended and well dispersed and, except where noted, triplicate aliquots from 300 Control triplicate flasks were taken for DNA and protein analyses. The determi nation of DNA was performed according to the method of Burton (7), and protein content was measured by the method of Lowry ef al. (32). Progesterone Receptors. The in situ receptor binding affinity of 250 progestins was assessed by their acute ability to translocate PR. Briefly, cells were incubated with progestins for 5 min at 37Â° as described previously (37). They were then harvested, cooled, and homogenized, and cytosols were prepared. These were incubated at 4Â°for 4 hr with 200 20 nM [3H]R5020 only or together with a 100-fokJ excess of unlabeled R5020 to measure nonspecific binding. After the incubation, unbound hormone was precipitated with dextran-coated charcoal, and aliquots of the charcoal-resistant supematants were counted to determine residual 150 cytoplasmic PR (21 ). Insulin Receptors. Harvested cells were washed twice in 50 ml binding buffer (minimal essential medium containing 1% bovine serum albumin, 25 mw Tricine, 25 mw 4-(2-hydroxyethyl)-1-piperazineethane- Â«100 sulfonic acid, pH 7.8 at 16Â°)and suspended at approximately 50 to 75 R5020 10-8M /:[) DNA/ml, and 1-ml aliquots of cells were used for insulin-binding studies or for DNA analysis (7). For insulin binding and Scatchard analysis, pelleted cells were resuspended in triplicate in 1 ml binding 50 buffer containing 125Hnsulin (0.3 or 0.4 ftg/m\) alone or together with unlabeled insulin (1 to 1000 ng/ml). For single point assays, nonspecific O binding was assessed in the presence of unlabeled insulin (50 /ng/ml). After incubation at 16Â°for 3 hr with gentle shaking, the reaction was 2 024 10 terminated by transfer of triplicate 200-//I aliquots/reaction mixture, to Treatment Time (days) prechilled 400-/

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Tabtel this study, and no effect was seen with other classes of steroid Effect ofprogestin concentration on growth inhibition and on PR translocationin hormones. We have tested several other synthetic progestins, cellsGrowthT47D0,human breast cancer including medroxyprogesterone acetate, megestrol acetate, and cytopiasmic PR (% ofcontrol)"R5020103 the antiprogestin RU38 486 (15); all suppress growth in T47Dco control)Hormone inhibition (% of cells. We have recently shown that, after brief (1 hr) progesterone one100 concentration0.1 treatment, PR translocate to nuclei, are down-regulated in a 11M 73 Â±1; 49 Â±4" processing step, and then replenish to control values within 24 1.0hM10.0nM0.1 87 Â±2 89 90 60 Â±2; 39 Â±3 85 Â±4 39 58 hr. In contrast, after a 1-hr pulse of R5020, replenishment is ItM 54 + 2Progesterone100Â±377 Â±3 8 38 blocked for at least 4 days (22). We therefore asked whether a 1.0 MMR5020102Â±2"'c 64Â±6Residual 3Progester 12 brief (1 hr) pulse of R5020 would have a long-term biological * Confluent flasks of T47D cells were treated for 5 min at 37Â°with the hormones ut the concentrations shown. Cytosols were then prepared from these cells. effect as measured by cell growth. Chart 3 shows a study in Cytoplasmic PR levels were determined and were compared to PR in untreated which cells were incubated 1 hr with medium containing R5020 controls. Receptor levels in controls were: R5020,12.4 pmol/mg DMA; progester and then washed extensively and reincubated with R5020-free one, 8.5 pmol/mg DNA. 6 Mean Â±S.E. medium for 14 days. Compared to that of hormone-free controls, 0 Cells from confluent flasks were split at a 1:5 ratio into duplicate 75-sq cm the growth of R5020-pulsed cells was suppressed for at least 4 flasks and treated every 2 days for 10 days with medium containing 5% charcoal- stripped fetal calf serum and the hormone concentration shown. On Day 10, days, after which the cells resumed growth at a rate similar to triplicate aliquota of cells from each flask were assayed for DNA and protein that of the controls. Other synthetic progestins are similarly long- content. DNA/protein ratios were constant so that cell size was unchanged. "Cells from confluent flasks were split at a 1:7 ratio into triplicate 25-sq cm acting (not shown). flasks and then treated and assayed as in Footnote c. Progestins and Insulin Receptors. Cell proliferation in culture is under complex regulation involving not only exogenous factors added to the growth medium but growth-stimulatory and inhibi tory factors that can be synthesized by the cells themselves. Control Insulin is a common medium supplement and is required for the 10'7MHÂ«râ€”10'8M10-8MHH10'8MH10'7MH10'7M10'7M10'7MMMH R5020

Estradici

Dihydrotestosterone 1 hour pulse, 1(T7M R5020 175 Testosterone

Dexamettiasone 150 Control, Hydrocortisone

1,25 COH)2Vitamin DS 125

17Â«(OH) Progesterone

100 200 300 400 500 600 100 ug DNA/Flask Chart 2. Steroid specificity of growth inhibition in T47D,Â»cells. Cells were plated in duplicate 75-sq cm flasks at a 1:5 split ratio (approximately 1.5 x 10* cells/flask) using stock growth medium. Forty-eight hr later, they were switched to medium 75 containing 5% charcoal-stripped fetal calf serum, with or without added hormones at the concentrations shown. Five days later, cells were suspended, and four 1-ml aliquots/flask were taken for DNA determinations. The average of 8 measurements is shown; bars, S.E. "p < 0.005. 50 hr between media changes, while R5020 is not metabolized under these conditions and is present continuously.5 All subse 25 quent experiments were therefore done with the nonmetaboliz- able progestin. Since progesterone and R5020 inhibit cell growth at concentrations that rapidly mobilize cytoplasmic PR, this response appears to be physiological and mediated by the 0246 10 12 14 Days After R5020 Removal receptors. Sparsely plated cells are particularly sensitive to the growth-inhibitory effects of R5020 (Table 1). Chart 3. Chronic effect of a 1-hr pulse of R5020 on T47D,Â«,proliferation. Cells were plated in 25-sq cm flasks at a 1:7 split ratio. Twenty-four hr later (Day 0), the The steroid specificity of growth inhibition is shown in Chart medium was replaced for 1 hr with medium containing either 10 7 M R5020 (â€¢)or 2. In this study, cells were split relatively densely and then treated hormone-free medium (O). The cell surface was then washed 3 times with RPMI, and the cells were reincubated in hormone-free medium for 14 days. Flasks were 5 days with a variety of hormones at high physiological concen fluid-changed every 48 hr. At the indicated time points, the cells from triplicate trations. Only R5020 had significant growth-inhibitory action in flasks were suspended in Ca^-Mg^-free Hanks'-EDTA, and triplicate 1-ml aliquots/ flask were taken for DNA analyses. Each point represents the average of 9 6 K. B. Horwitz, unpublished data. determinations; bars, S.E.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1985 American Association for Cancer Research. GROWTH REGULATION BY PROGESTINS continuous serum-free growth of all human breast cancer cell lines in which it has been tested (2-4). Moreover, insulin and 100 steroid hormones can reciprocally regulate each others' actions 80 oCJoIO (8, 28, 38, 42). The present studies show that progestins are 60 OC coÂ° among the heterologous hormones that regulate insulin receptor Ã¨,C\JLLI levels. Chart 4 shows that T47Dco express insulin receptors on 40 \-I their surface and that the receptors can be regulated by R5020. rÃ¤ ^I i This study compares insulin receptors in cells grown 96 hr in .y o 20-20 insulin-free medium or in cells grown in the same medium but supplemented with 0.1 MM R5020. The competitive binding w curves and Scatchard analyses of the binding (Chart 4, inset) are shown. Assuming a negative cooperative model for the analysis -40-60-80100Ã¬f-o!t- of the curvilinear Scatchard plots, the increased binding seen 3OXOQ after 96 hr of treatment with R5020 is consistent with an increase in the number of insulin binding sites per cell and no change in binding affinity. Progestin treatment increased the number of insulin receptors from approximately 8,000 to about 18,000 sites/cell. Chart 5. Steroid specificity of insulin receptor modulation. Duplicate 75-sq cm To determine the hormone specificity of the increase in insulin flasks were plated at a 1:5 split ratio, grown for 2 days, and then switched to medium containing charcoal-stripped serum with or without (control) the hormones receptors, cells were incubated for 5 days with several classes at the concentrations shown. Treatment continued for 5 days with daily fluid of steroid hormones or analogues, at concentrations sufficient changes. Cells from each flask were harvested separately, 4 aliquots were taken to bind and translocate their respective receptors but below for DMA analysis, and triplicate aliquots/flask were used for assay of specific insulin binding as described. Binding was normalized to DMA and is shown as percent of pharmacological levels (Chart 5). The increase in insulin receptors control insulin receptors (1.6%/100 /jg DNA); bars, S.E. Â£2.estradiol; DHT, dihydro was specific to R5020; neither estradici nor dihydrotestosterone testosterone; Dex, dexamethasone; RU, RU38 486. had any effect, consistent with reports in other systems. The synthetic glucocorticoid dexamethasone decreased the number receptors (or other proteins) may be a useful marker to distin of insulin binding sites by 50%. Glucocorticoids are known to guish progestins from antiprogestins in T47Dco. Other hormones modulate insulin receptor levels, and their effects are variable, that had no effect on insulin receptors were 1,25-dihydroxyvitamin D3 (10~7 M), testosterone (10~8 M), 21-hydroxyprogester- depending on the cell type and the conditions used in the studies. RU38 486, a synthetic steroid with antiglucocorticoid and anti- one (10~7 M), and the antiestrogen nafoxidine (10~6 M). progestin actions (15), had no effect on insulin receptor levels Receptors for insulin and insulin-like growth factors are known when it was added to the cells alone (Chart 5); together with to vary with cell growth rate (46). We therefore investigated the R5020, it partially blocked receptor induction by R5020 (not relationship between insulin receptors and growth, using un shown). We are testing the possibility that regulation of insulin treated and R5020-treated cells (Chart 6). Untreated T47DÂ«,cells treated with 10 or with 100 nw R5020 for 1 to 9 days were harvested and assayed for insulin receptor levels and for total DNA content. In untreated cells, the number of insulin binding sites/cell rose slightly during the first round of cell division (Days 1 to 3) and then settled down to control levels. In contrast, during the first 3 days, when little if any growth was demonstrable in R5020-treated sets, the number of insulin binding sites/cell rose 4- to 5-fold and peaked on Days 2 to 3. Thereafter, slow cell R5020 10'7M proliferation was paralleled by a fall in insulin receptor levels to 0.1 0.3 0.5 0.7 0.9 about twice control values, where a new steady state was Insulin Bound tng/100ug DNA) established.

DISCUSSION Control It is extremely difficult to understand the mechanisms of tumor regression produced by endocrine therapies. A variety of treat ment methods that influence many different hormones are all 10 100 1000 about equally effective in generating remissions (36). The com Insulin Concentration (ng/ml) plexities involved in the regulation of hormones and their recep Chart 4. Effect of R5020 on 125l-insulinbinding to cultured 1470Â«,human breast tors, by both homologous and heterologous hormones, virtually cancer cells. Cells were grown for 4 days in the presence (O) or absence (â€¢)of 10~7 M R5020. Triplicate flasks were harvested, cells were washed, and aliquots defy explanation in vivo and are almost as difficult to sort out in vitro. Variant and mutant cells with deletions of key regulatory of the cells were taken for DNA analysis and for duplicate insulin binding assays as described in "Materials and Methods." Binding data were normalized to DNA components resulting in hormone resistance can be used to content. Competition curves: each point represents the average of 6 experimental points (bars, S.E.), and specific 1Z5l-insulinbound is plotted against insulin concen simplify analysis of such complex interrelationships, and we have tration. Scatchard plots (inset): the ratio of bound (B) to free (F) hormone is plotted used a variant human breast cancer cell line with this strategy in as a function of the total insulin bound; both are normalized to 100 ^g DNA. mind. In progesterone target tissues, including breast cancer,

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o Insulin Receptors DNA OÃe a. 5 500 o o i Â§ 4 400 o m a-

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ER+. PRÂ± Clin. Endocrinol.Metab., 50: 246-250,1980. 7. Burton, D. A. A study of conditions and mechanisms of the diphenylamine reaction for the colorimetrie estimation of deoxyribonucleicacid Biochem J Jam 62:315-323,1956. 8. Butler, W., Kelsey, W. H., and Green, N. Effects of serum and insulin on the sensitivity of the human breast cancer cell line MCF-7 to estrogens and antiestrogens. Cancer Res., 41: 82-88,1981. * It Antiproliferative 9. Chalbos,D., and Rochefort, H. Dualeffects of the progestin R5020on proteins released by the T47D human breast cancer cells. J. Biol. Chem 259-1231- 1238,1984. 10. Chalbos, D., Vignon, F., Keydar, I., and Rochefort, H. Estrogensstimulate cell proliferation and induce secretory proteins in a human breast cancer cell line (T47D).J. Clin. Endocrinol.Metab., 55: 276-283,1982. 11. Clark, G. M., McGuire, W. L, Hubay, C. A., Pearson, 0. H., and Marshall, J. S. Progesteronereceptors as a prognostic factor in Stage II breast cancer N ER-, PR* Engl. J. 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A mode) describing possible rotes of progestins in the treatment of nisms of action. In: K. Fotherby and S. B. Pal(eds.),Hormones in Normal and breast cancer. See the "Discussion"for details. Tarn,tamoxifen; Prog, progestin. Abnormal HumanTissues, pp. 81-113. New York: W. de Gruyter, 1981. 17. Horwitz, K. B., and Alexander, P. S. In situ photolinked nuclear progesterone receptors of human breast cancer: subunit molecular weights after transfor between the antiestrogen and the progestin could result in loss mation and translocation. Endocrinology, 773: 2195-2201,1983. of PR altogether, leaving a progestin-insensitive cell population 18. Horwitz, K. B., and McGuire,W. L. Progesteroneand progesteronereceptors in experimentalbreast cancer. Cancer Res., 37:1733-1738,1977. (11). 19. Horwitz, K. B., and McGuire,W. L. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1985 American Association for Cancer Research. Growth Inhibition and Increase of Insulin Receptors in Antiestrogen-resistant T47D co Human Breast Cancer Cells by Progestins: Implications for Endocrine Therapies

Kathryn B. Horwitz and Gary R. Freidenberg

Cancer Res 1985;45:167-173.

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