Estrogen Receptor Expression Activates the Transcriptional and Growth-Inhibitory Response to Retinoids Without Enhanced Retinoic Acid Receptor a Expression1

Home , Retinoic acid receptor, Retinoid receptor, Retinoid X receptor

[CANCER RESEARCH 58, 5110-5116. November IS. 1998] Estrogen Receptor Expression Activates the Transcriptional and Growth-inhibitory Response to Retinoids without Enhanced Retinoic Acid Receptor a Expression1

Angelika Rosenauer, Clara Nervi, Kelly Davison, William W. Lamph, Sylvie Mader, and Wilson H. Miller, Jr.2

Lady Davis Institute for Medical Research. SMBl) Jewish General Hospital. Mcuill University. MontrÃ©al.Quebec H3T IE2. Canada Â¡A.K., K. D., W. H. M.Â¡;Dipartimento di Istologia timi Embriologia Medica. UniversitÃ di Roma "!M Sapienza." 14,00161 Rome, Italy Â¡C.N.I; Ligand Pharmaceuticals. San Diego, California 92121 Â¡W.W. L.]; and UniversitÃ©de Munirent. MontrÃ©al.QuÃ©bec,H3C 3J7 Canada /S. M.Â¡

ABSTRACT present in the promoter regions of target genes (7). The receptors are divided into two categories: RARs and RXRs, each composed of three Estrogen receptor (KR)-positive human breast cancer cells are hormon- receptor subtypes (a. ÃŸ,and y). RARs have a high affinity for both ally regulated and are inhibited by retinoids, whereas most ER-negative breast cancer cells arc not. Here, we compared retinoid-induced tran- tRA and 9cisRA. a naturally occurring retinoid that also binds and scriptional activation and growth inhibition in the ER-negative breast activates RXRs (8, 9). It is interesting that the response to retinoids in cancer cell line \1DA-MB-231, stably transfected to express wild-type ER breast cancer cell lines appears to be correlated with the expression of (S3Ãœ),with that of the ER-positive MCE-7 line and the ER-negative ER. ER-positive cells are. in general, growth-inhibited by retinoids, parental line. Retinoids inhibited growth of the ER-expressing S30 clone whereas ER-negative cells are not (5, 10-12). but not of the parental MDA-MB-231 cells. Unlike a previously reported Previous reports have shown that ER-positive HBC cell lines ex MDA-MB-231 subclone that was transfected to express a mutated ER press higher levels of RARa (12, 13). Furthermore, an MDA-MB-231 (G40ÃœV),S30 did not express increased levels of retinoid receptor RNA or clone stably transfected with a mutated form of ER (G400V.HEO) had protein, nor was there increased binding activity to retinoid-responsive a restored ability to respond to the growth-inhibitory effects of RA, as DNA elements. However, stable expression of ER increased retinoid acti well as increased RARa mRNA expression (14). We tested whether vation of transcription of a retinole acid (RA) response elements from the low level in MDA-MB-231 to approach the level of MCF-7. The restored an increased level of RAR expression was responsible for the restored retinoid response of S30, a subclone of the ER-negative breast cancer growth inhibition and transcriptional regulation by RA were unaffected cell line MDA-MB-231 (clone 10A) that is stably transfected to by treatment with ER agonists or antagonists. Transient expression of ER but not of other nuclear receptors in MDA-MB-231 cells also activated constitutively express wild-type ER (G400.HEGO) at levels similar to retinoid-induced transcription, showing that this response is specific to those found in MCF-7 (10. 15). Unexpectedly, we found that expres ER. Furthermore, the effect of exogenously expressed ER on retinoid sion of ER in this subclone of MDA-MB-231 has activated retinoid- response was much greater than that obtained by overexpression of RA induced transcription and regulation of gene expression without receptor a and/or retinoid X receptor a. Finally, a panel of ER mutants changing baseline RAR levels. Furthermore, transient expression in showed that enhancement of retinoid-induced transcriptional activity was MDA-MB-231 of wild-type ER directly stimulated the transcriptional dependent on the integrity of the DNA binding domain. response to RA. Importantly, this activation was greater than that obtained by transfection of RAR, RXR, or RAR combined with RXR. We further explored this effect by transfection of other nuclear hor INTRODUCTION mone receptors, as well as ER constructs deleted in various functional Retinoids are a group of natural and synthetic vitamin A derivatives domains. that play important roles in normal cellular growth and differentiation of human tumor cell lines in vitro ( 1). There is evidence that retinoids may induce differentiation and inhibit growth of HBC1 (2, 3). RA has MATERIALS AND METHODS been shown to inhibit growth of many but not all breast cancer cell Cell Culture. S30 cells, obtained courtesy of Dr. V. C. Jordan (Northwest lines in vitro and in athymic mice (4-6). Retinoids mediate their ern University. Chicago, IL), were routinely cultured in a-MEM phenol effects by binding to a group of nuclear receptors belonging to the red-free medium (Life Technologies, Inc., Burlington, Ontario, Canada) sup superfamily of nuclear receptors that includes ER. The activated plemented with 5% charcoal-stripped serum (16) and 500 /ng/ml G418 (Life retinoid receptors are transcription factors that bind to RAREs that are Technologies. Inc.). Early-passage MCF-7 cells were obtained from the Amer ican Type Culture Collection (Manassas. VA) and maintained in a-MEM plus phenol red (Life Technologies, Inc.) in 5% PCS (Upstate Biotechnology Inc., Received 6/24/98: accepted 9/17/98. Lake Placid, NY). MDA-MB-231 (clone 10A; a gift from Dr. V. C. Jordan) The cosls 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 cells were cultured in a-MEM plus phenol red (Lite Technologies, Inc.) and 18 U.S.C. Section 1734 solely lo indicate this fact. 5% PCS (Upstate Biotechnology Inc.). All media were supplemented with 100 1This work was supported by the Cancer Research Society, McGill Center for Translalional Research; by Fonds pour la Formation de Chercheurs et l'Aide Ã la units/ml penicillin and 100 fig/ml streptomycin and maintained at a pCO2 of 5% in 37Â°Chumidified air. Recherche; by the London Life Award in Medical Research; and. in pan. by the Associa/ione Italiana per la Ricerca sul Cancro. W. H. M. Â¡sa Scholar of the Medical S30 growth experiments were performed by culture of 10,000 cells per well Research Council of Canada, S. M. is a recipient of a Chercheur Boursier award from the in the above medium at the indicated concentrations of reagents: tRA and E2 Fond de Recherche en SantÃ©de Quebec, and K. D. is supported by a Fonds de la Recherche en SantÃ©du Quebec/Fonds pour la Formation et l'Aide Ã la Recherche (Sigma Chemical Co., St. Louis, MO); 9cisRA. kindly provided by Dr. R. A. Heyman (Ligand Pharmaceuticals. San Diego, CA); and ICI 164,384. kindly studentship. : To whom requests for reprints should be addressed, at Lady Davis Institute for provided by Dr. A. E. Wakeling (Zeneca, Macclesfteld, United Kingdom). All Medical Research. 3755 Chemin de la Cote-Ste-Catherine. MontrÃ©al.QuÃ©becH3T 1E2. reagents were replenished every 3 days, and quadruplicate samples were Canada. Phone: (514)340-8222 ext. 4365: Fax: (514)340-7576: E-mail: WMillerts1 counted on days 3, 5. and 8 by hemocytometer. Trypan blue cells never LDI.JGH.mcgill.ca. *The abbreviations used are: HBC. human breast cancer; RA. retinoic acid: TR. exceeded 1% and were excluded from the count. Controls for hormone-treated thyroid hormone receptor: ER. estrogen receptor; RARE. RA response element: RAR. RA cells were treated with identical concentrations of vehicle alone, which did not receptor. RXR, retinoid X receptor: IRA. all-Ãra/iÃ-RA;9cisRA. 9-c/j-RA; E2, 17/3- exceed 0.02% (v/v) of the media. eslradiol: OAPDH. glyceraldehyde-3-phosphate dehydrogenase: nt. nucleotide(s): HPLC. Northern Analysis. Cells were washed twice in PBS and extracted with high-performance liquid chromatography; CAT, chloramphenicol acetyltransferase; guanidine thiocyanate to prepare total RNA (17). RNA samples were electro- (3-gal. ÃŸ-galaclosidasc: RE. response clement; PR. progesterone receptor; AR. androgen receptor: DBD, DNA binding domain; LBD. ligand binding domain: ERE. estrogen phoresed on a 1% formaldehyde agarose gel and blotted onto a Zeta probe responsive element. transfer membrane (Bio-Rad Laboratories, Mississauga. Ontario, Canada). The 5110

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. ESTROGEN RECEPTOR RESTORES RETINOIC ACID RESPONSE obtained filters were hybridized to cDNA probes labeled by random priming ICI 164,384. As reported previously, E2 alone inhibited growth of (Pharmacia Biotech, Baie D'Urte, QuÃ©bec,Canada). Hybridizations were per these cells (Ref. 15; Fig. 1). When ICI 164,384 was added, this formed according to the manufacturer's specifications (Bio-Rad). Hybridization growth-inhibitory effect was abolished. As shown in Fig. l, IGT5 M probes were isolated from plasmids containing: a 0.6-kb Pstl fragment of the tRA caused a 73% growth inhibition of these cells, which was not human RARa cDNA (18); a 1.8-kb Ea>Rl fragment of the human RXRa cDNA significantly altered by the addition of ICI 164,384. (19); a 0.56-kb Pstl fragment of the pS2 cDNA (American Type Culture Collec Retinoid Receptor Expression and Regulation in ER-positive tion); and a 1.2-kb Pstl fragment of GAPDH (American Type Culture Collection). and ER-negative HBC Cells. Because previous reports have corre RNase Protection Assay. Total RNA was isolated as described above and lated RAR levels with the increased response to RA of ER-expressing the assay performed according to Zinn et al. (20). Hybridization of cRNA probes was carried out at 45Â°Covernight, followed by the addition of 300 Â¿ilofRNase HBC cells (12-14), we compared RAR expression levels in the wild-type digestion buffer containing 40 /ig/ml RNase A and 700 units/ml RNase Tl. RNase ER-expressing S30 line with its parental MDA-MB-231 line and the digestion was performed at 25Â°Cfor 1 h. The RNase-resistant fragments were naturally ER-positive MCF-7 cells. Fig. 2A shows RNase protection resolved by electrophoresis on 6% urea-polyacrylamide sequencing gels. To analysis examining the expression of RARs in these cells grown with or control for RNA loading, we included a GAPDH probe in all samples. As without IO"6 MtRA treatment. RARa, RARy, and RXRa expression in approximate size markers, "P-labeled Afa/il-digested fragments of pBR322 were the MCF-7 cell line was found to be down-regulated by RA treatment run on all gels. The RNA protection probes for each of the five human retinoid (Fig. 1A and Table 1). Neither RARa nor RXRa levels of expression receptor isoforms (both RARs and RXRs) were PCR-amplified from cloned differed between S30 and MDA-MB-231 cells, and both cell lines ex sequences as follows: RARa, nt 1797-1922 (21): RARÃŸ,nt 1545-1721 (22); pressed lower levels of these receptors than did MCF-7. Furthermore, RARy. nt 415-554 (23); RXRa, nt 1760-1854 (19); and RXRÃŸ,m 1610-1739 (24). The resulting fragments were cloned into pGEM4Z (Promega). and their these receptors were not found to be regulated by RA. In the absence of identity was confirmed by nt sequencing. The specificity of each probe for human RA treatment, RAR/3 expression was only detected in the parental ER- sequences was verified by testing the probe against both mouse and human RNAs negative cell line. However, RA treatment induced a modest expression known to contain corresponding mRNA. A probe corresponding to GAPDH was of the RARÃŸmessage in both MCF-7 and S30 cells. ER expression in purchased from Ambion Inc. (Austin, TX). S30 seems to be associated with basal inhibition but restored retinoid RA Binding Assay. Cells (1-5 X IO8) were rinsed twice in PBS and induction of RARÃŸexpression, suggesting that the presence of ER is collected by trypsinization. Nuclear extracts were prepared as described pre viously (25) and incubated for 18 h at 4Â°Cwith 10 nM [3H]tRA (52 Ci/mmol; linked to the control of RARÃŸinducibility by RA. RARy expression was found in MCF-7, but it was not found in MDA-MB-231 or its ER- DuPont NEN Research Products, Boston, MA) in the presence or absence of a 200-fold molar excess of tRA. The extracts were fractionated at 4Â°Cby positive subclone. The lack of RARy expression in S30 and MDA-MB- 231 cells differs from the results by Roman et al. (13). However, clonal HPLC over a SuperÃ³se 12 HR 10/30 size exclusion column (Pharmacia, selection of MDA-MB-231 has been reported to result in the differential Uppsala. Sweden) at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were recovered, and the radioactivity was determined using a scintillation counter. expression of RAR subtypes (28). This may explain our inability to detect Transient Transfections and CAT Assays. MCF-7, MDA-MB-231 the RARy message in MDA-MB-231 cells, which were clonally selected (1 X IO6), and S30 (0.7 X IO6)cells were plated in 35-mm dishes and allowed to and from which S30 cells were derived (15). No variation was seen in attach overnight in regular growth medium. Transfections were performed by RXRÃŸexpression among the cell lines (Fig. 2A and Table 1), and RXRy washing the cells once with Opti-MEM (Life Technologies. Inc.), then adding a was not expressed. mixture of Lipofectamine reagent (Life Technologies, Inc.) and 0.3 fig of a Because we did not find RAR RNA levels increased in the ER- ÃŸREtkCATreporter gene (a gift from Dr. H. Sucov. Institute for Genetic Medi positive transfectant, we asked whether expression of RAR protein cine, University Southern California School of Medicine, Los Angeles, CA) that carries the sequence 5'-GGTTCACCGAAAGTTCACTCG-3'. with 0.3 /ig of differed among the three cell lines by evaluating ligand binding capacity. Nuclear extracts obtained from untreated MCF-7, MDA- CMVÃŸgalplasmid (Clontech Laboratories, Palo Alto, CA) as an internal control. MB-231. and S30 cells were incubated with [3H]tRA in the absence For transfections of MDA-MB-231 cells with expression vectors, cells were plated at a density of 4.3 X IO6cells in 60-mm plates per treatment group and cotransfected with a 1:5:5 ratio of expression vector to ÃŸREtkCATand CMVÃŸgal.The transfection was stopped after 5 h by the addition of phenol red-free medium and 5% charcoal-stripped serum. Cells were harvested 48 h posttransfection, and the 200- ÃŸ-galactivity was assayed (26). The CAT activity was measured according to a modified protocol of the organic diffusion method (27). Cell extracts were stan dardized to ÃŸ-galactivity in a total of 50 /xl of 0. l MTris-Cl (pH 7.8) and incubated at 37Â°Cwith 200 /xl of 1.25 mM cold chloramphenicol (ICN. Costa Mesa, CA) and 0.25 ju,Ci of 3H-labeled acetyl-CoA (NEN; Streetsville, Ontario, Canada). The reaction was allowed to proceed for 4 h, after which the organic phase was extracted with Ready Organic Scintillation Cocktail (Beckman. Mississauga, On tario, Canada). The radioactivity of 750 Â¿Â¿Iofthe organic phase was counted on a beta counter (Beckman).

RESULTS

Retinoid Response Is Independent of ER Stimulation or Inhi bition. The cell line S30, derived by stable transfection of the ER- negative MDA-MB-231 (clone 10A) transfected with wild-type ER (HEGO), is growth-arrested in the presence of 10~6-10~5 M tRA 1 23456789 days In culture (10). We investigated whether this effect is dependent on ligand binding to ER, as reported for an MDA-MD-231 clone transfected to Fig. 1. Growth response of the ER-positive transfectant S30 cell line to tRA and E2 in the express mutated ER (HEO; Ref. 14). In this experiment, 10~s M tRA presence or absence of the antiestrogen ICI 164.384. S30 cells were plated at IO4cells per well in charcoal-stripped phenol red-free medium. Hormones and inhibitor, at the concentrations was used to obtain maximal reversible inhibition of cell growth. In described in the legend, were replenished every 3 days. Data [tinnt-i. cell counts of four Fig. 1, S30 cells were cultured for 8 days in the presence of 10~5 M independent wells. Trypan blue-positive cells never exceeded 1% and were excluded from the count. SEs (not shown) were, in all cases. < 10% of cell numbers. â€¢¿media:â€¢¿tRA(10~5 tRA or 10~8 M E2, with or without 10~7 M pure estrogen antagonist M):A. IRA/ICI (10~5-10~7 M),Q ICI ( 1CT7M);O, E2 (10"* M);A. E2/IC1 (1(T*-10~7 M).

5111

or presence of a 200-fold excess of unlabeled tRA, and the binding of [3H]tRA to specific receptors was determined (Fig. 2B). As reported RARs RXRs previously (11), the HPLC profile of nuclear extracts prepared from 1234 56 789101112 I 2 3 4 5 6 7 8 9101112 MCF-7 cells showed two major peaks with specific [3H]tRA binding

RXRy - activity eluting with retention times of 25 and 31 min (11). The peak RARÃŸ- at 31 min, seen only in MCF-7 nuclear extracts, corresponds to a protein of M, 18,000 and may represent contamination by cellular RA-binding protein from the cytosol. The peak at 25 min observed in i? all three cell types indicates the presence of the A/r 50,000 RA-binding RARs. The RA-binding profiles are consistent with the RNA analysis RARy- and show that S30 cells have comparable binding activity to MDA- MB-231 but lower binding activity than MCF-7. Gene Regulation in ER-positive Cell Lines. Because it has been RARo- reported that estrogen may regulate RAR levels in ER-expressing cells (14, 29, 30), we asked whether the low expression in S30 would be altered upon estrogen stimulation. We compared RARa and RXRa expression in MCF-7 and S30 cell lines when they were cultured with and without 10~8 M E2 (Fig. 3/4). Estrogen stimulation in S30 or

QAPOH- MCF-7 did not increase the transcript levels of RARa or RXRa. Although RXRa is regulated in MCF-7 by retinoids (Refs. 10 and 31; B Table 1), S30 cells showed no retinoid regulation of RARa or RXRa expression (Table 1). Retinoids have been shown to regulate expres MCF7 sion of estrogen-dependent genes in MCF-7 cells but not in MDA- 15 - MB-231 cells, in which they are not expressed (10, 11). Because expression of ER in S30 reactivates expression of pS2, we asked whether RA can regulate this gene. As shown in Fig. 3B, pS2 gene expression decreases in the presence of 10~6 M tRA and is almost 10 undetectable after 3 days. This loss of pS2 expression persists after 6 days (data not shown). Retinoid-induced Transcriptional Activation in ER-positive 5 - and ER-negative Cell Lines. Because expression of wild-type ER in S30 cells restores some RA-regulated gene expression, we asked whether it alters the ability of the MDA-MB-231 parental cell line to co O mrrrrrrrrrnj ^ support transcription of RA-responsive promoters and whether this response differs from that of MCF-7 cells. We transiently transfected I MDA 231 MCF-7, S30, and MDA-MB-231 cells with a thymidine kinase-CAT I 15 reporter driven by the RARE of the RARÃŸ2promoter (DR5). As shown in Fig.4/4, tRA and 9cisRA strongly induced transcription from this promoter element in MCF-7 and S30 cells but only weakly induced transcription in MDA-MB-231 cells. We further tested a B 10 TREpal RE in these cell lines. Induction by 10~6 M tRA gave a 3.8-fold increase of transcription in MCF-7 cells, whereas S30 and MDA-MB-231 cells showed a minimal 1.3-fold increase in transcriptional activation (data not shown). MDA-MB-231 and S30 cells express lower levels of RAR protein than do MCF-7 cells, and RARa overexpression has been shown to I 0 confer responsiveness to RA (32). We, therefore, tested whether the transcriptional activity of MDA-MB-231 cells could be restored to the S30 level of S30 and MCF-7 cells by overexpression of RAR or RXR. Fig. 15 - 4B shows MDA-MB-231 cells transiently cotransfected with DR5 and

10 - Fig. 2. Retinoid receptor expression and regulation in ER-positive and ER-negative cell lines. A, RNase protection analysis for retinoid receptor expression of 20 Â¿tgoftotal RNA isolated from MCF-7 (Lanes 1-4), S30 (Lanes 5-7). and MDA-MB-231 (Lanes 9-12) cells. Lanes 1. 5. and 9. medium alone; Lanes 2, 6. and 10, medium containing vehicle; Lanes 3, 7. and //, I0~6 M IRA treatment for 1 day; Lanes 4. 8. and 12. IO"6 M tRA treatment for 3 days. The RNase-protected bands corresponding lo the specific receptors are identified, and GAPDH expression was used as a quantitative loading control. B. RA-binding profile in the three cell lines. Size-exclusion HPLC analysis of [*H]RA binding in nuclear extracts of MCF-7 (top). MDA-MB-231 (middle), and S30 (bottom) cells grown in optimal medium. Extracts from each cell line were incubated with 10 nM (3H]tRA in the absence (â€¢)or presence (O) of a 200-fold excess of unlabeled tRA. 0 10 20 30 40 50 Extracts were subjected to HPLC analysis using a SuperÃ³se 12 HR 10/30 size exclusion Retention time (min) column with a recovery of 90-95% total protein. The radioactivity in each fraction was determined. 5112

Table I uensiumielric results of Â¡heKNase protection analysis"

typeS30dl7.04.0NE'2.26.2NEd38.24.82.48.4C12.014.02.56.2MDA-231di10.817.5NE2.48.4NEd315.011.54.07.2

RARaRARpRARTRXRâ€žRXRâ€žRXR,,C"24.0012.89.86.1MCF-7dl23.0012.55.95.8NEd38.96.23.81.89.0C8.502.86.9Cell

" Data shown are represented in arbitrary units, obtained by phospoimaging analysis of the gel in Fig. 2A. The value for each sample was normalized for RNA loading by using the GAPDH signal. Treatments were as follows: C. medium containing vehicle alone: dl. tRA treatment for I day; d3. tRA treatment for 3 days. ' NE. not expressed. receptor types as indicated. Increased RARa or RXRa expression did increase transcriptional activity over that of tRA alone. In contrast, a not stimulate an increase in transcriptional activity on the DR5, combination of RA and E2 added a 2-fold increase in transcriptional although overexpression of both gave a 3-fold increase in transcrip activity when the mutant receptor was present. This result confirms tional induction. However, transient cotransfected with HEGO and the the results of Sheikh et al. (14) but indicates that the wild-type DR5 element was performed, a large induction was observed. Clearly, receptor does not require activation by its own ligand for maximal transient expression of the wild-type ER was able to increase tran transcriptional activity on a DR5 element. Furthermore, HEGO trans- scription on this promoter element above that seen with RARa or fected MDA-MB-231 cells showed the retinoid-induced transcrip RXRa alone or both in combination. Thus, this activation of ER is tional activity was not significantly decreased in the presence of ICI unlikely to be solely mediated by increased RAR activity. Further (Fig. 4B). Thus, the transcriptional effect of the wild-type ER was more, this increased transcriptional activity by RA is seen in the stable independent of ER stimulation or inhibition. ER transfectant and, transiently, in the parental cell line, suggesting Because overexpression of RAR and RXR was less effective at that the integration site of ER in the S30 genome is not involved in activating RA-dependent transcription than the expression of ER. we producing this effect. asked whether other steroid hormone receptors that are not highly We further asked whether this activation of RA-induced transcrip expressed in MDA-MB-231 cells could substitute for the effect of ER tion was independent of ER stimulation. Sheikh et al. (14) reported on RARE activation. The choice of other steroid hormone receptors that the transcriptional activity of a stable HEO-transfected MDA- used was guided by an extensive study performed by Hall et al. (33), MB-231 cell line on a DR5 element, when stimulated by tRA, was who found that ER-negative HBC cell lines did not express the PR or enhanced upon addition of estrogen. We examined in a transient the AR. Fig. 5 compares the abilities of the above receptors, the TR, transfection assay whether estrogen could also increase the transcrip and wild-type ER (HEGO) to support transcriptional activity on the tional activity on this RARE in the presence of the wild-type ER. Fig. DR5 element. We found that none of these receptors was able to 4C shows the result of transiently cotransfected MDA-MB-231 cells activate transcription from the RARE to the level of the wild-type ER, with either the wild-type ER (HEGO) or the mutant receptor (HEO) suggesting that a unique relationship exists in these cells between and the DR5 element. In the presence of the wild-type receptor, RA-induced gene activation and the presence of the ER. addition of 10~8 M E2 in combination with 10~6 M tRA did not To further define this relationship and to begin to determine its

MCF-7 830

Fig. 3. Analysis of gene regulation in MCF-7 and S30 cells. A. Northern blot analysis of the expression â€”¿RXRaâ€”¿ of the RARa and RXRa message when ER-positive cells were stimulated by estrogen. Cells were grown in steroid-stripped phenol red-free medium with or without 10~" ME2 for the number of days indicated. â€”¿RARaâ€”¿ E2 was replenished every 3 days. Ten /ig of total cellular RNA were probed with either RXRa or RARa cDNA probes, as indicated for each panel. GAPOH Control lanes represents cells treated with vehicle 6h 24h 3d 6d alone. The corresponding GAPDH expression is 10-"ME2 represented beneath each panel. B. RA-induced reg control control IO* M E2 ulation of pS2 gene expression in S30 cells. Cells were treated with 10~6 M tRA for the time periods indicated. Ten Â¿tgoftotal cellular RNA were loaded per lane. Control lanes represent cells treated with vehicle alone. The transcript corresponding to the B) pS2 gene is identified. GAPDH expression, as in A, pS2 is represented beneath each panel.

GAPDH - fin. 2d 3d fin Id. 10-*MtRA control 5113

mechanism, we examined which functional domain(s) of the ER receptor are required for RA-responsiveness in these cells. We per formed transient transfections using selected ER deletion mutants and tested their transcriptional activity on the DR5 element in MDA-MB- 231 (Fig. 6). The HE15 construct lacks the COOH-terminal LBD (AF2), (HE619) contains a deletion of the NH2-terminal A/B domain (AF1), and HE11 is missing the core of the DBD (34). The HE82 construct was generated by introducing three point mutations in the zinc finger motif (CI finger region) of the DBD, causing it to recog nize a glucocorticoid RE but not the ERE (35). Both HE11 and HE82 were constructed to express the wild-type ER LBD. Fig. 6 demon strates that deletion of the DBD (HE11 ) or a changed RE recognition (HE82) virtually abolished the activation of transcription of the DR5 element, whereas neither the NH2-terminal nor the ligand binding deletion of ER reduced the transcriptional activity observed with wild-type ER (HEGO). Statistical analysis using a two-tailed t test MDA231 showed no significant differences in transcriptional activity between HEI l and the vector controls pSG5 and pKCR2, whereas significant differences were found between HEGO, HE 15, and HE 19 when they B were compared to vector controls (P < 0.001). These results suggest that the DBD of ER plays a key role in the response of these cells to RA-induced transcription.

DISCUSSION ER-negative breast cancer cells demonstrate a variety of features of increased malignancy compared to ER-positive cells, including more frequent genetic changes and increased invasive and metastatic po tential, as well as lack of response to hormonal therapy (35). The stable expression of ER in an ER-negative cell line, thus, provides a model to determine which differences in malignant phenotype can be attributed to the loss of ER alone. The S30 cell line expresses ER at the level seen in MCF-7 cells (15), restoring ligand inducibility of some ER target genes. Although expression of ER did not reconstitute estrogenic regulation of cellular proliferation, it did restore the ability MDA RAR RXR RAR/RXR HEGO HEGO/ICI of retinoids to inhibit growth. This suggests that ER expression is co-transfected receptors responsible for the differential response to retinoids of ER-positive versus ER-negative cells. This growth inhibition has also been re ported with a mutant ER (HEO; Ref. 14) with altered ligand binding affinities (36, 37). The importance of ER expression is confirmed by transient transfection of wild-type ER, which significantly stimulated RA-dependent transcription of the J3RE retinoid RE. Because transiently expressed genes are not integrated into the genome, this finding provides strong evidence against the possibility that the integration site of ER into the genome of S30 influences the observed activation of retinoid re sponse. The specificity of the ER effect on retinoid-induced gene activation was demonstrated by the fact that neither the transiently expressed ER-responsive PR nor the related AR or TR was able to transactivate the RARE in MDA-MB-231 to the same level as wild-

experiments performed in triplicate; bars. SE. B. fold induction by retinoids in MDA- MB-231 cells when they were cotransfected with receptors (0.06 u.g) as indicated. 0.3 fig HEGO HEO of the ÃŸREtkCATreporter construct, and 0.3 jig of CM VÃŸgal.Columns, activation by the indicated retinoids at IO"" M in MDA (no cotransfected receptor). RARa (RAR). RXRa tRA(10"*M) + - + + - + (RXRÃŒ.their combination (RAR/RXR), and wild-type ER with (HEGO//CÃ•) or without (HEGO) 10~7 M ICI 164.384. Cells were grown in steroid-stripped phenol red-free E2 (10-7M) - + + + + medium. Columns, represents results from three independent transfections; bara, SE. C, fold induction by IRA and estrogen in MDA-MB-231 cells transiently transfected with 0.06 u,g of either wild-type ER (HEGO) or mutant ER (HEO) on DR5-mediated tran Fig. 4. ReCinuid-induccd (ranscriptional activation in ER-positive and ER-negative cell scription. ÃŸREtkCAT (0.3 ^g). and CMVÃŸgal (0.3 /ig). Cells were grown as in B. lines. A. Fold induction by 10 6 MIRA or 9cisRA of CAT expression from a ÃŸREtkCAT- Columns, results from two independent transfections performed in triplicate; bars, SE. For conlaining reporter construct in MCF-7, S30. and MDA-MB-231 cells. Cells were grown all of the above experiments, cells were harvested 48 h posttransfection. Cell extracts were in their preferred medium and transfected with 1 /Ag of the ÃŸREtkCATreporter plasmid normalized with respect to /3-gal activity. Fold induction was calculated by dividing the and I jig of CMVÃŸgal expression vector. Columns, results from three independent value in cells treated with 10 " Mretinoids by the value in cells that received vehicle only. 5114

type ER. This suggests the existence of a unique relationship between ER expression and the retinoid response in HBC cell lines. One possible explanation tor the increased sensitivity to retinoids of ER-positive cells is an ER-dependent increase in the level of retinoid nuclear receptors, particularly RARa (14, 29, 30). Consistent with these reports, we found that RAR expression at the level of RNA and that ligand binding activity is higher in MCF-7 than in the ER- negative MDA-MB-231 cell line. Furthermore, MDA-MB-231 cells stably expressing a mutated form of ER (HEO; Ref. 14) have been reported to express more RARa than their ER-negative parental line. None However, in the transfected S30 line, we found both the RNA level 140 of RARa (and its binding partner. RXRa) and the nuclear RA-binding fold Induction capacity unchanged from the MDA-MB-231 parental cell line. We Fig. 6. Effect of ER deletion mutants on transcriptional activation of 0KARE. Fold further determined that increased levels of RARa could not restore induction by IO " M IRA in MDA-MB-231 transiently transfected with KR deletion retinoid-induced transcriptional activation of an RARE in MDA-MB- mutants. The structuro lor each expression vector of the ER deletion constructs is indicated. W. DBD: LBD (AF2) and NH,-terminal (Al-1) domains are marked. *. three 231 to the level seen in the transfected S30 line or in MCF-7 (Fig. 4fi). point mutations in this region (see text). A total of 0.06 /ig of expression vector. 0.3 jig Thus, the level of RAR expression is not the limiting factor in the of ÃŸREtkCAT. and 0.3 /ig of CMVÃŸgal were used per transfection. with pSG5 and transcriptional response to RA. Our results suggest additional mech pKCR2 representing vector controls. Cells were grown in charcoal-stripped phenol red-free medium. Shown are HEGO (wild-type ERl. HF.I1 (DBD deletion). HKH2 (DBD anisms are involved in the ER-dependent effect on retinoid response. point mutationsl. HEI5 (LBD deletion). HEIV (A/B deletion), and pSG5 and Â¡>KCK2 The HEO-transfected MDA-MB-231 clone described by Sheikh el (vector controls). Cells were transfected. and fold induction was calculated as in Figs. 4 al. (14) expressed more RARa than its parent, but this difference was and 5. Columns, means of a minimum of four independent transfections performed in triplicate; bars. SE. only apparent after estrogen stimulation. In S30. stimulation by es trogen did not elevate RA-receptor transcript levels, nor was the increased retinoid activity of this cell line dependent on ligand acti of the DR5. Furthermore, ICI 164.384 was not able to inhibit the vation of ER. Sheikh ft til. (14) also demonstrated that maximal HEGO-mediated increase in RA dependent transcription (Fig. 4fl) or activation of transcription of an RARE by expression of HEO required influence growth inhibition in S30 cells in the presence of RA (Fig. 1), the presence of estrogen. Consistent with these data. Fig. 4C shows suggesting that ER transcriptional activity on EREs is not involved. that transient transfection of MDA-MB-231 with HEO increased the ER could affect several different levels by which the RA response can ability to support transcription on an RARE in the presence of estro be regulated, such as RAR-DNA binding or postbinding transcriptional gen. However, we did not observe this effect with transfection of the activity. A potential for cross-talk between ERs and RARs may exist due wild-type ER. and estrogen conveys no effect on RA-induced gene to the recognition of shared half-sites in their REs, as has been described activation in S30 cells. These functional differences may be related to previously (41. 42). Furthermore, ER may modulate expression or avail altered conformation of the mutant ligand binding domain. Indeed, the ability of coregulators of RA-dependent transcription. Several potential unliganded wild-type ER is localized mainly in the nucleus, whereas coregulators of RA-activated transcription have been reported recently HEO is found mainly in the cytoplasm in association with hsp90 and (43-52). Furthermore, restored transcriptional activity in S30 may be requires the binding of ligand for nuclear translocation (38-40). stimulated by ER expression, leading to activation of retinoid receptors, Functional analysis of ER mutants suggest that the DBD of ER is the absolute abundance of which has not changed. This might be medi important in activating the RA response (Fig. 6). Deleting the DBD or ated by differences in phosphorylation of RARs between MDA-MB-231 altering its recognition from an ERE to a glucocorticoid response and S30. Reports of studies performed both in vivo and HI vitro have element-like RE abolished the ER-induced transcriptional activation shown that RA-dependent phosphorylation of RARs increases binding to and transcriptional activity on RAREs (53-56). It is possible that one or more of these above possibilities may 120-1 result in the differential response of ER-positive and ER-negative HBC cells to retinoids. We have previously shown that retinoids lower 100 H ER protein levels in HBC cells, and others have shown synergistic inhibition of mammary tumors in mice by combined treatment with 80- retinoids and antiestrogens. In patients with-breast cancer, hormonal treatment is limited by the development of resistance to tamoxifen and other ER-inhibitory ligands. In some patients, this progression has 60- I been linked to mutations in the ER LBD (57). Our findings, consistent with those of Sheikh et al. (14), show that mutations or deletions in 40- the ER LBD that alter or eliminate the inhibitory activity of tamoxifen do not affect the ability of ER to stimulate retinoid response. Thus, 20- retinoids might have activity even in some cancers that have lost hormone dependence. Understanding the mechanisms linking ER with the retinoid response in HBC cells may lead to new combined hor pSGS HEGO PR AR TR monal approaches to treat or prevent HBC. co-transfected receptors

Fig. 5. Transcriptional activation on a RARE of selected steroid hormone receptors when compared with wild-type ER. Fold induction by IO "M IRA on ÃŸRARE-mediated ACKNOWLEDGMENTS activation in MDA-MB-231 cells transiently transfecled with 0.06 fig of expression vectors pSG5 (control), wild-type F.R (HEGO}. PR. AR. and TR. 0.3 /ig of jaREtkCAT. We thank Dr. Henry Sucov for kindly providing (he /SREtkCAT REs; Dr. and 0.3 fig CMVÃŸgal.Cells were grown in steroid-stripped phenol red-free medium and Pierre ChambÃ³n for the RARa. AR. and PR expression vectors; and Dr. John harvested 48 h posltransfection. Fold induction was calculated as described in Fig. 4. White tor making the TR expression vector available. 51 15

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Angelika Rosenauer, Clara Nervi, Kelly Davison, et al.

Cancer Res 1998;58:5110-5116.

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