The Key to the Antiestrogenic Mechanism of Raloxifene Is Amino Acid 351 (Aspartate) in the Estrogen Receptor1

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[CANCER RESEARCH 58. 1872-1875, May I. 19981 The Key to the Antiestrogenic Mechanism of Raloxifene Is Amino Acid 351 (Aspartate) in the Estrogen Receptor1

Anait S. Levenson and V. Craig Jordan2

Robert H. Lurte Comprehensive Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611

ABSTRACT action of raloxifene can be obtained by comparing and contrasting the biology of wild-type ER with mutant forms of the receptor. We have The crystallization of the ligand-binding domain (LBD) of the estrogen previously identified a natural mutation of the ER in a tamoxifen- receptor (ER) with 17ÃŸ-estradiol and raloxifene [A. M. Brzozowski et al., stimulated breast tumor grown in athymic mice (9). Using single- Nature (Lond.), 389: 753â€”758,1997] now provides a molecular basis for stranded Â«informational polymorphism and sequencing, we identified the biological activity of complexes as either agonists or antagonists. It is well established that the critical structural feature of antiestrogens is a a single point mutation at AA 351 where aspartate was replaced by correctly positioned alkylaminoethoxy side chain. The X-ray crystallog tyrosine. Additionally, another mutant is available with an AA sub raphy clearly shows that the alkylaminoethoxy side chain of raloxifene stitution in approximately the same region. A cloning error produced causes a specific and inappropriate molecular perturbation of the LBD a mutant ER, in which a glycine is replaced by valine at position 400 and that the nitrogen in the side chain must hydrogen bond with aspartate (Ref. 10; Fig. 2). 351 in the LBD of ER. Our goal in this paper was to test the hypothesis that the AA 351 is We previously identified and characterized a naturally occurring mu the key to the antiestrogenic activity of raloxifene. To accomplish the tation in the ER from a tamoxifen-stimulated transplantable human goal, we have compared and contrasted the pharmacology of ralox breast tumor line. The mutation is at AA351 of LBD, where the aspartate ifene to block or induce estrogen-stimulated increase of TGF-a is changed to tyrosine (Asp351Tyr). mRNA in MDA-MB-231 breast cancer cells stably transfected with In this report, we compared and contrasted the pharmacology of raloxifene to block or induce l\,-stimulated increase in TGF-a mRNA in cDNA for the wild-type or mutant ERs (Gly400Val and Asp351Tyr; stable transfectants of ER-negative human breast cancer cells with the Refs. 11 and 12). cDNAs from wild-type, mutant-amino acid (AA) 400 ER and mutant-AA 351 ER. Our results show that the mutation at AA 351 that replaces MATERIALS AND METHODS aspartate by tyrosine specifically changes the pharmacology of raloxifene from an antiestrogen to an estrogen. By contrast, a mutation at AA 400 Cell Culture. The three stable transfectants (S30, ML-a-2H, and BC-2 cell does not, and the antiestrogenic properties of raloxifene are retained. lines) used in this study were constructed from the ER-negative MDA-MB-231 These data and the fact that the nitrogen in the side chain must specifically (clone 10A) human breast cancer cells as described previously (11. 12). These interact with aspartate 351 makes this the key to the antiestrogenic cells stably express either the wild-type ER (S30 cells; Ref. 11), the Gly400Val activity of raloxifene. mutant ER (ML-a-2H cells; Ref. 11), or the Asp351Tyr mutant ER (BC-2 cells; Ref. 12). Cells were maintained in phenol red-free MEM with 5% charcoal-dextran treated calf serum, supplemented with 100 units/ml penicil INTRODUCTION lin, 100 /xg/ml streptomycin, 2 mM L-glutamine. 6 ng/ml bovine insulin, 100 The key structural feature of the nonsteroidal antiestrogens is a mM nonessential AAs. and 500 /Â¿g/mlG418. All of the tissue culture solutions correctly positioned alkylaminoethoxy side chain ( 1). Alterations in were from Life Technologies. Inc. (Gaithersburg, MD). E2 was purchased from Sigma Chemical Co. (St. Louis, MO), raloxifene was obtained from Eli Lilly the distance between the nitrogen and oxygen atoms (2, 3), restrictions Laboratories (Indianapolis. IN), and ICI 182.780 was a gift from Dr. Alan in the movement of the side chain (4), or alterations in the basicity of Wakeling (Zeneca Pharmaceuticals, Macclesfield, United Kingdom). All com the nitrogen (5) all cause changes in antiestrogenic activity. Further pounds used in these experiments were dissolved in 100% ethanol and added more, removal of the side chain causes a change in the pharmacology to the medium in 1:1000 dilution for a final concentration no higher than 0.2%. of the molecule from an antiestrogen to an estrogen or results in the Northern Blot Analysis. RNA extraction and Northern blot analyses were complete loss of biological activity (6). performed essentially as described previously (13). Briefly, total RNA was Recently, the crystal structure of the LBD3 of the ER was deter isolated from cells following 24-h treatment with compounds using the Trizol mined with either E-, or the antiestrogen raloxifene in the ligand reagent (Life Technologies). Twenty fig of RNA sample were fractionated in a 1.2% agarose-formaldehyde gel and transferred to a nylon membrane binding site (Ref. 7; Fig. 1, A and B). As anticipated, the backbone of (Nybond-N +; Amersham Corp.. Arlington Heights, IL). The membranes were raloxifene concisely binds to the appropriate AAs that lock E-, into the hybridized at 42Â°Cwith 12P-labeled TGF-a probe (plasmid generously pro LBD (7). As predicted (8), the alkylaminoethoxy side chain projects vided by Dr. Rik Derynck. Genentech. Inc.. South San Francisco. CA). The out of the hydrophobic pocket. However, the interesting observation is membranes were then washed and autoradiographed by exposure to Hyperfilm that the nitrogen in the side chain binds to AA aspartate at position (Amersham) at â€”80Â°Cwith intensifying screens for 2-3 days, and the ex 351 (Fig. IB). pected 4.8 kb transcript was detected in all three cell lines. Subsequently, the Clearly proof of the essential role of AA 351 in the antiestrogenic blots were stripped and reprobed with ÃŸ-actin cDNA. The signals were quantitated by Phosphorlmager (Molecular Dynamics) analysis (Image Quant Received 1/6/98; accepted 3/4/98. software). The costs of publication of this article were defrayed in pan 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 faci. RESULTS 'These studies were supported by NIH Grant CA-56143 and by the continued generosity of the Lynn Sage Breast Cancer Foundation of Northwestern Memorial Regulation of Endogenous TGF-a mRNA. We have previously Hospital. 2 To whom requests for reprints should be addressed, at Robert H. Lurie Comprehen demonstrated that there is a concentration-dependent induction of sive Cancer Center. Northwestern University Medical School. 303 East Chicago Avenue. TGF-a mRNA by E2 in MDA-MB-231 cells stably transfected with 8258 Olson Pavilion. Chicago. IL 60611. Phone: (312) 908-4148: Fax: (312) 908-1372. cDNAs from wild-type and mutant ER (13, 14). In the present study, ^ The abbreviations used are: LBD. ligand-binding domain: ER. estrogen receptor: AF. transcriptional activation function; E2, 17ÃŸ-estradiol;4-OHT. 4-hydroxytamoxifen: AA, we compared and contrasted the ability of E2, raloxifene, and the pure amino acid: TGF-a, transforming growth factor a. antiestrogen ICI 182.780 to regulate TGF-a mRNA in the transfec- 1872

cells. However, the pure antiestrogen ICI 182,780 was an antiestrogen, and blocked the action of raloxifene in all cell lines. Antiestrogenic .Side Chain

DISCUSSION

We have used a specific bioassay in situ, the estrogen regulation of the TGF-a gene to test the hypothesis that a precise mutation at AA HV Ilis 524 351 of ER can affect the pharmacology of the receptor-raloxifene Asp 351 v complex. The crystal structure of the ER-raloxifene complex shows that there is a key interaction of the alkylaminoethoxy side chain with AA 351 (7). Our results demonstrate that the mutation at AA 351 that replaces aspartate by tyrosine specifically changes the pharmacology of raloxifene from an antiestrogen to an estrogen (Fig. 5). By contrast, \ a mutation at AA 400 does not, and the antiestrogenic properties of // NH Arg 394 raloxifene are retained. The results from this bioassay now provide Glu 353 important evidence about the key pharmacological role of AA 351. The side chain creates an inappropriate perturbation of the LBD of the wild-type ER so that the whole orientation of the complex prevents gene transcription. The X-ray crystallography of E2 with the ER LBD B (7) demonstrates that the steroid is enveloped in a pocket that closed by helix 12. Apparently, this is critical for the recruitment of coactivators to the AF-2 site and the initiation of RNA polymerase activity. Estradici The antiestrogen raloxifene causes a change in the orientation of helix 12, so that it is repositioned and now cannot recruit coactivalors. During the past few years, there has been a considerable effort to describe the biological relevance of the AF-2 region through site- directed mutagenesis (15-17). The AAs 538, 542, and 545 are known

) ;O to be essential for AF-2 activity and gene transcription (Fig. 2; Refs. 18 and 19). However, antiestrogen can become more estrogenic if Glu 353 HO' there are mutations at straddling AAs [e.g., L543A/L544A or M547A/ Water L548A (20) or L540Q (21); see Fig. 2]. It would seem that the Arg 394 mutations permit the recruitment of some coactivators even at the Fig. 1. The interaction of raloxifene (Al and E, (ÃŸ)inthe LBD Ã¶l'theER adapted from surface of the inappropriately positioned helix 12. Be that as it may, Ref. 7. it is clear that the critical AA that provokes all of the other changes is Asp351. A mutation at AA 351 prevents the specific interaction with the side chain and does not allow the conformational change associ tants so that we could evaluate our results in the light of the known ated with antiestrogen action. crystal structure for the ER complex (7). The effects of compounds on These data not only define the mechanism of action of raloxifene the expression of TGF-a mRNA in S30, ML-a-2H, and BC-2 cells but also illustrate, for the first time, a specific mechanism of drug were determined by Northern blot analyses 24 h after the addition of resistance to antiestrogens. Tamoxifen is effective as a long-term compounds alone or in combinations. Although the results from adjuvant therapy to provide survival advantages in node-positive and multiple experiments are combined in Fig. 4, the data shown in Fig. node-negative breast cancer (22). However, the clinical data point to 3 illustrate that E2 stimulates accumulation of TGF-a mRNA in all the eventual emergence of tamoxifen-stimulated tumor growth (23, three cell lines. Raloxifene and ICI 182.780 did not cause an increase 24). In the laboratory, tamoxifen-stimulated tumors have been devel in TGF-a mRNA levels in either the S30 or ML-a-2H cell lines, and oped by the long-term treatment of athymic mice inoculated with both compounds blocked the action of E2. In contrast, raloxifene did MCF-7 breast cancer cells (25, 26). The tumors retain ER and will increase TGF-a mRNA in BC-2 cells, and unlike ICI 182,780, ralox grow in response to either tamoxifen or E2 (26). The class of drugs ifene was not an inhibitor of E2 action. As shown in Fig. 4, raloxifene known as pure antiestrogens inhibit tumor growth (27), and prelimi was an estrogen only when using BC-2 cells and not the ML-a-2H nary clinical evidence suggests that the pure antiestrogen ICI 182,780

Ligand Binding Domain AF-1 AF-2

Fig. 2. Schematic representation of the wild-type human ER cDNA. The positions for single point mutations Gly400Val and Asp351Tyr are indicated. The AF-2 region and various mutant receptors generated by random chemical- or site-directed mutagenesis arc shown that either ABCD545, E 542l cause loss of AF-2 activity (538, 542, and 545; Ref. 19) or cause an 538,302 increase in estrogenic activity if the receptor is liganded with an antics- trogen (540. 543. 544. 547. and 54Ã„;Refs. 20 and 21). 540 'B 400 543 1 The key to the antiestrogenic >- 351 544F553 548

1873

The TGF-a gene is known to be estrogen regulated, and we discov ered that estrogen increases the transcription of TGF-a mRNA in our transfectants (14). The present results confirm and extend our previous study (13) and TGFa determine that a specific mutation in a critical region of the ER from S30 a tamoxifen-stimulated breast tumor can enhance the estrogenic prop (wt ER) erties of an antiestrogen. The cells with a mutant receptor would have ÃŸ-Actin a growth advantage. For the future, the crystal structure of the 4-OHT-ER complex is required to compare and contrast antiestrogens. On the basis of data we have recently published with our transfectants (32), we predict that ML-U-2H the shape of the 4-OHT-ER complex will be significantly different (mutant400ER) from the raloxifene-ER complex. We have noted that 4-OHT can increase TGF-a mRNA in both wild-type and mutant transfectants, although the mutant Asp351Tyr ER complex appears to be more efficacious (32). The wild-type ER-4-OHT complex is clearly more promiscuous than the raloxifene-ER complex, so the helix 12 con BC-2 taining the AF-2 AAs must be available for ER-protein interactions in (mutant,,, ER) a context replete with coactivators. This simple molecular model would explain the reduced uterine estrogenicity of antiestrogens of the raloxifene class and their ability to block the uterotrophic properties of Fig. 3. Effects of E2 and antiestrogens or combinations of compounds on TGF-a mRNA expression in ER transfectunts analy/ed by Northern blot as described in "Mute- tamoxifen (33, 34). Additionally, and perhaps more importantly, it rials and Methods." The sources for RNAs were the following: Commi, cells treated with points to an explanation for the primary mechanism of drug resistance elhanol vehicle; Â£2.cells treated with 1(T" ME, (S30 cells) and with 10 * ME, (MLaZH to tamoxifen. Tamoxifen-stimulated growth may be driven by selec and BC-2 cells); Ral, cells treated with 10"" Mraloxifene; ICI, cells treated with IO"6 M ICI 182.780; E2 + Kai. cells treated with I0~v or 10~8 M E2 (depending on the cell line) tion of cells that have high levels of coactivators. The selection and 10"'' Mraloxifene; E2 + ICI. cells treated with IO"9 or IO"8 ME, (depending on the process could be enhanced with an ER with a mutation in the AA 351 cell line) and I0~6 M ICI 182.780; Ral + ICI. cells treated with 10~" M raloxifene and region, but this would not be obligatory for tamoxifen-stimulated IO"6 M ICI 182.780. ÃŸ-ActinmRNA was used as a loading control. growth. The model would then predict that raloxifene would not, in is an effective second-line therapy following tamoxifen failure (28, 29). Antiestrogenic Estrogenic For the past 10 years, we have been particularly interested in the mechanism of tamoxifen-stimulated growth (30). One potential hy pothesis we investigated was the random appearance of a mutant receptor that would interpret an antiestrogen as an estrogen. This would provide a growth advantage for breast cancer cells and ulti Deflected mately produce a tamoxifen-stimulated tumor. The mutant receptor. Asp351Tyr. was identified as the major mRNA species (80%) in a tamoxifen-stimulated tumor. However, rather than test the biological activity of the mutant in transient transfection assay using yeast or Tyr 351 animal cells, we chose to prepare stable transfectants in the ER- negative human breast cancer cell line MDA-MB-231 (11, 12). Our rationale was that, given that human ER was involved in the growth process in breast cancer, it would function optimally in the context of Wild Type Receptor Mutant Receptor breast cancer cells replete with coactivators. Similarly, we chose to Fig. 5. The key to anliestrogen activity of raloxifene. The replacement of Asp by Tyr identify a gene target in situ rather than use artificial reporter genes at AA 35 1 creates an inappropriate interaction at the LBD of the ER and prevents the shift with optimal estrogen response elements derived from animals (31). of the AF-2 site to an antiestrogenic conformation.

B wt ER D mutant400ER â€¢ nHItant, MER Fig. 4. Induction of TGF-a mRNA expression in ER transfec tants. The graph shows the inducible levels (the ratio of normal ized TGF-a mRNA levels in cells treated with E2 or anliestro- gensrnormali/ed TGF-a mRNA levels in untreated cells) of TGF-a mRNA as determined by densitometric analyses of the autoradiograms The mean of inducible TGF-a mRNA level from three independent experiments is represented. The concentrations mentioned in the legend to Fig. 3 for each compound were used alone and in combination experiments.

E2 Ral ICI E2+1CI E2+Ral Ral+ICI 1874

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. ANTIESTROOENIC MECHANISM OF RALOX1FENE general, be completely cross-resistant with tamoxifen and could po 17. Zhuang, Yâ€žKatzenellenbogen. B. S.. and Shapiro. D. Estrogen receptor mutants tentially be used on tamoxifen failure. which do not hind 17/3-estradiol dimerize and bind to the estrogen response clement in vivo. Mol. Endocrinol.. 9: 457-566. 1995. 18. Danielian, P. S.. White. R.. Lees. J. A., and Parker, M. G. Identification of a ACKNOWLEDGMENTS conserved region required for hormone dependent transcriptional activation hy steroid hormone receptors. EMBO J., //: 1025-1033. 1992. 19. Tzukerman. M. T.. Esty, A.. Santiso-Mere. D.. Danielian. P.. Parker. M. G., Stein, We thank Henry Muenzner for preparing schematic figures. R. B.. Pike. J. W.. and McDonnell. D. P. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. The Key to the Antiestrogenic Mechanism of Raloxifene Is Amino Acid 351 (Aspartate) in the Estrogen Receptor

Anait S. Levenson and V. Craig Jordan

Cancer Res 1998;58:1872-1875.

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