A Novel Antiestrogenic Mechanism in Progesterone Receptor-transfected Breast Cancer Cells

The expression of progesterone receptor (PR) is normally estrogen-dependent, and progesterone is only active in target cells following estrogen exposure. This study revealed that the effect of estrogen was markedly disrupted by estrogen-independent expression of PR. Transfection of PR in estrogen receptor (ER)-positive MCF-7 cells abolished the estradiol-17β growth stimulatory effect that was observed in the parental cells and the vector-transfected controls in a ligand-independent manner. The antiestrogenic effect was also observed at the level of gene transcription. Estradiol-17β (E2)-induced gene expression of pS2 and GREB1 was impaired by 50–75% after 24–72 h of E2 treatment in PR-transfected cells. Promoter interference assay revealed that PR transfection drastically inhibited E2-mediated ER binding to estrogen response elements (ERE). The antiestrogenic effects of transfected PR are associated with enhanced metabolism of E2. HPLC analysis of [3H]E2 in the samples indicated that the percentage of [3H]E2 metabolized by PR-transfected cells in 6 h is similar to that by vector-transfected control cells in 24 h (77 and 80%, respectively). The increased metabolism of E2 may, in turn, be caused by increased cellular uptake of E2, as demonstrated by whole cell binding of [3H]E2. The findings open up a new window for a hitherto unknown functional relationship between the PR and ER. The antiestrogenic effect of transfected PR also provides a potential therapeutic strategy for estrogen-dependent breast cancer. The expression of progesterone receptor (PR) is normally estrogen-dependent, and progesterone is only active in target cells following estrogen exposure. This study revealed that the effect of estrogen was markedly disrupted by estrogen-independent expression of PR. Transfection of PR in estrogen receptor (ER)-positive MCF-7 cells abolished the estradiol-17β growth stimulatory effect that was observed in the parental cells and the vector-transfected controls in a ligand-independent manner. The antiestrogenic effect was also observed at the level of gene transcription. Estradiol-17β (E2)-induced gene expression of pS2 and GREB1 was impaired by 50–75% after 24–72 h of E2 treatment in PR-transfected cells. Promoter interference assay revealed that PR transfection drastically inhibited E2-mediated ER binding to estrogen response elements (ERE). The antiestrogenic effects of transfected PR are associated with enhanced metabolism of E2. HPLC analysis of [3H]E2 in the samples indicated that the percentage of [3H]E2 metabolized by PR-transfected cells in 6 h is similar to that by vector-transfected control cells in 24 h (77 and 80%, respectively). The increased metabolism of E2 may, in turn, be caused by increased cellular uptake of E2, as demonstrated by whole cell binding of [3H]E2. The findings open up a new window for a hitherto unknown functional relationship between the PR and ER. The antiestrogenic effect of transfected PR also provides a potential therapeutic strategy for estrogen-dependent breast cancer. Ovarian steroid hormones estrogen and progesterone are essential for the normal growth and development of the breast. The two hormones also play critical roles in the regulation of breast cancer development. It has been established that estrogen stimulates the growth of breast cancer cells both in vivo and in vitro (1McGuire W. Carbone P. Vollmer R. Estrogen in Human Breast Cancer. Raven Press, New York1975Google Scholar, 2Jensen E.V. Cheng G. Palmieri C. Saji S. Makela S. Van Noorden S. Wahlstrom T. Warner M. Coombes R.C. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15197-15202Crossref PubMed Scopus (193) Google Scholar, 3Gottardis M.M. Wagner R.J. Borden E.C. Jordan V.C. Cancer Res. 1989; 49: 4765-4769PubMed Google Scholar). Antiestrogens have been the front-line therapy for hormone-dependent breast cancers (4MacGregor J.I. Jordan V.C. Pharmacol. Rev. 1998; 50: 151-196PubMed Google Scholar), which are estrogen receptor (ER) 1The abbreviations used are: ER, estrogen receptor; ERE, estrogen response element; E2, estradiol-17β; PR, progesterone receptor; PR-A, PR isoform A; PR-B, PR isoform B; eiPR, estrogen-independent PR; RT, reverse transcription; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; HPLC, high performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; Ab, antibody. - and progesterone receptor (PR)-positive. Nonetheless, antiestrogen-induced remissions are often followed by acquisition of antiestrogen resistance and ultimately disease relapse (5Clarke R. Liu M.C. Bouker K.B. Gu Z. Lee R.Y. Zhu Y. Skaar T.C. Gomez B. O'Brien K. Wang Y. Hilakivi-Clarke L.A. Oncogene. 2003; 22: 7316-7339Crossref PubMed Scopus (393) Google Scholar). The acquired resistance is mostly manifested by insensitivity to antiestrogens such as tamoxifen and increased ER activity (6Berstein L.M. Zheng H. Yue W. Wang J.P. Lykkesfeldt A.E. Naftolin F. Harada H. Shanabrough M. Santen R.J. Endocr. Relat. Cancer. 2003; 10: 267-277Crossref PubMed Scopus (45) Google Scholar). It has been shown that signaling via the epidermal growth factor receptor and HER-2/neu can activate both ER and the important ER coactivator AIB1 (7Osborne C.K. Schiff R. Breast. 2003; 12: 362-367Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Breast tumors with high levels of AIB1 and HER-2 may be resistant to tamoxifen because of an increase in its estrogen agonist activity. The development of strategies for the effective treatment of tamoxifen-resistant breast cancer is one of the main challenges for breast cancer research. Whereas it is established that estrogen stimulates the growth of breast cancer cells, the function of progesterone in breast cancer remains controversial. Progestins were found to stimulate growth, have no effect, or inhibit growth depending on the experimental conditions and the status of hormone receptors (8Sutherland R.L. Hall R.E. Pang G.Y.N. Musgrove E.A. Clarke C.L. Cancer Res. 1998; 48: 5084-5091Google Scholar, 9Groshong S.D. Owen G.I. Grimison B. Schauer I.E. Todd M.C. Langan T.A. Sclafani R.A. Lange C.A. Horwitz K.B. Mol. Endocrinol. 1997; 11: 1593-1607Crossref PubMed Scopus (229) Google Scholar, 10Jeng M.H. Parker C.J. Jordan V.C. Cancer Res. 1992; 52: 6539-6546PubMed Google Scholar, 11Lin V.C. Ng E.H. Aw S.E. Tan M.G. Ng E.H. Chan V.S. Ho G.H. Clin. Cancer Res. 1999; 5: 395-404PubMed Google Scholar). This controversy reflects our insufficient understanding of progesterone biology and has hampered effective applications of progestins or antiprogestins in breast cancer treatment. The controversies over the effect of progesterone in breast cancer are due to several complexities in the PR system. One of the complexities is that PR is an estrogen receptor-dependent gene product (12Druege P.M. Klein H.L. Green S. Stack G. Chambon P. Ryffel G.U. Nucleic Acids Res. 1986; 14: 9329-9337Crossref PubMed Scopus (47) Google Scholar, 13Graham J.D. Roman S.D. MaGowan E. Sutherland R.L. Clarke C.L. J. Biol. Chem. 1995; 270: 30693-30700Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and the action of progesterone requires priming treatment of estrogen to induce PR. It is conceivable that the prior presence of estrogen may significantly confound assessment of the role of progesterone on growth and other cellular processes in breast cancer cells. On the other hand, the effects of progesterone also depend on a complex interaction between estrogen, progesterone, and their receptors. Studies have demonstrated the suppression of estrogen-stimulated ER activity by agonist- and antagonist-occupied PR (14Kraus W.L. Weis K.E. Katzenellenbogen B.S. Mol. Cell. Biol. 1995; 15: 1847-1857Crossref PubMed Scopus (175) Google Scholar, 15Katzenellenbogen B.S. J. Soc. Gynecol. Investig. 2000; 1: S33-S37Crossref Google Scholar). However, ER can transmit signals received from the agonist-activated PR to the Src/p21 (ras)/Erk pathway (16Migliaccio A. Piccolo D. Castoria G. Di Domenico M. Bilancio A. Lombardi M. Gong W. Beato M. Auricchio F. EMBO J. 1998; 17: 2008-2018Crossref PubMed Scopus (523) Google Scholar), suggesting a synergistic interaction between the ER and PR. Our previous work has shown that estrogen-independent expression of PR by transfection in the ER- and PR-negative breast cancer cells MDA-MB-231, facilitated a striking inhibition of cell growth by progesterone in vivo and in vitro (11Lin V.C. Ng E.H. Aw S.E. Tan M.G. Ng E.H. Chan V.S. Ho G.H. Clin. Cancer Res. 1999; 5: 395-404PubMed Google Scholar, 17Lin V.C. Eng A.S. Hen N.E. Ng E.H. Chowdhury S.H. Clin. Cancer Res. 2001; 7: 2880-2886PubMed Google Scholar). Progesterone also induced remarkable focal adhesions in the PR-transfected MDA-MB-231 cells (18Lin V.C. Ng E.H. Aw S.E. Tan M.G. Ng E.H. Bay B.H. Mol. Endocrinol. 2000; 14: 348-358Crossref PubMed Scopus (53) Google Scholar, 19Kastner P. Krust A. Turcotte B. Stropp U. Tora L. Gronemeyer H. Chambon P. EMBO J. 1990; 9: 1603-1614Crossref PubMed Scopus (1339) Google Scholar). This present study reveals that estrogen-independent expression of PR in MCF-7 cells exhibited strong antiestrogenic and antiproliferative effect that is independent of PR ligands. The antiestrogenic effect is associated with a marked decrease of estradiol-17β (E2) in the culture medium of PR-transfected cells. This suggests that the transfected PR exerted the antiestrogenic effect by modulating cellular uptake and metabolism of E2. The study provides the basis for a novel antiestrogenic mechanism that may be used for breast cancer treatment. Chemicals—All tissue culture reagents were from Invitrogen. Propidium iodide (PI), progesterone, 17β-estradiol, estrone, estriol, 6α-hydroxyestradiol, and 16α-hydroxyestrone were purchased from Sigma. Trichloroacetic acid was purchased from Fisher Scientific. Nonidet P-40 was purchased from USB Corp. (Cleveland, OH). [2,4,6,7,16,17-3H]Estradiol-17β (∼149 Ci/mmol) was from Amersham Biosciences. Cell Culture—MCF-7 cells were obtained from the American Type Culture Collection in 1995 at passage 147. Cells were routinely maintained in phenol red-containing Dulbecco's modified Eagle's medium supplemented with 7.5% fetal calf serum (Hyclone, Logan, UT), 2 mm glutamine, and 40 mg/liter gentamicin. For all experiments involving cell culture, phenol red-free medium containing 5% dextran-coated charcoal-fetal calf serum (DCC-FCS), and 2 mm glutamine was used, and this medium will be referred as Test Medium in the subsequent description. Transfection—PR expression vectors hPR1 and hPR2 contain human PR cDNA coding for PR isoform B and A, respectively, in pSG5 plasmid (18Lin V.C. Ng E.H. Aw S.E. Tan M.G. Ng E.H. Bay B.H. Mol. Endocrinol. 2000; 14: 348-358Crossref PubMed Scopus (53) Google Scholar). Vector pBK-CMV (Stratagene) containing the neomycin-resistant gene was cotransfected with hPR1 and hPR2 into MCF-7 cells using in medium containing were for pSG5 by using for Cells transfected with both pBK-CMV and pSG5 (Stratagene) were used as transfection were from the cells by of and in containing and containing of were on and to Human PR and B were with from human was with from human by was used as Cell cells were in Test the cells were with E2 in medium from in which a of of controls received The medium with the was 2 and cells were with a Cell were in Test Medium for h were with E2 or progesterone for the indicated of cells were with in mm mm mg/liter and Nonidet for h in the The cells were in with of The were by for cell were from of was with Green on an The for pS2 are and The for GREB1 are and which for human was used as control for the of cDNA for gene were in and was The of from was on the basis of Promoter were in Test Medium for h were transfected with 2 of response or 2 of control h cells were medium and with E2 or as Cells were at and h after E2 treatment. activity was using the of were on in Test Medium for h were with [3H]E2 in phenol red-free Dulbecco's modified Eagle's medium for uptake was by [3H]E2 in the presence of of E2. the cells were with and and cellular [3H]E2 was with of and by a liquid of by were in for h were with [3H]E2. The were following and 24 h treatment. with [3H]E2 were used as controls for The in the were with acid followed by with The of the medium was to with HPLC was on a at The B The was as A; a increase to B; a increase to B; a increase to B; B. The was were and the was on a liquid treatment with to cell and growth were by analysis of followed by the of gene gene and E2 between control and cells were by of PR the levels of PR and ER in the parental cells the vector-transfected control cells and in PR-transfected cells and and cells PR MCF-7 and cells in the of E2. cells as as PR-A, as as the expression of estrogen-independent PR E2 was to induce PR in PR-transfected cells. also revealed that the ER was by following E2 treatment in all the cell On the other hand, PR transfection the levels of ER in the presence or of E2. PR-transfected MCF-7 cell and were for their to progesterone and The MCF-7 parental cells and the vector-transfected MCF-7 as control cells. E2 Cell in PR-transfected MCF-7 Cells after h of in 2 was to the effect of progesterone on cell in MCF-7 cells transfected with PR. The cells been with or E2 for 24 h were with and of progesterone for The of E2 treatment is The treatment was to induce PR that the effect of progesterone can be between PR-transfected cells and the control cells. The revealed that increase of PR by transfection in MCF-7 cells the action of progesterone on cell Whereas no at progesterone at an increase of the in both PR-transfected cells and and control cells and However, the of E2 on PR-transfected MCF-7 cells is the of that on control cells. with the effect, E2 increased the of MCF-7 and vector-transfected control cells by an of of the presence or of E2 cells by an of in PR-transfected cells and This effect of transfected PR is ligand-independent as progesterone at this antiproliferative effect of E2 in PR-transfected MCF-7 cells. PR Transfection in MCF-7 Cells the of that the of MCF-7 and cells were that of controls after the cell of and cells were the as that of cells. the cell of PR-transfected cell to be that of control cells after in culture, this an increased of cell growth in PR-transfected cells. shown by the of PR-transfected cells control is similar to that of parental and vector-transfected cells the effect of E2 on cell as a function of Cells were with as control or E2 for were and for cell by are as effect of of E2 on cell Cells were with as control or E2 for of and for cell Medium was with E2 at and h after the treatment. are as of PR Transfection at the of is a and the effect of E2 is by ER its gene regulation transfected PR the effect of E2 by the activity of ER, the expression of two estrogen target pS2 and GREB1 were by analysis E2 induced the increase of pS2 expression by after 24 h of treatment in all cell This of pS2 gene expression was in MCF-7 and cells after and h of treatment. and the of pS2 gene expression by E2 was impaired by 50–75% after and h of treatment GREB1 expression following and h of treatment was impaired by with that at the 24 h in PR-transfected cells. the of gene expression in MCF-7 and cells for both pS2 and GREB1 similar at all It is to that the inhibition of activity of ER by PR transfection at 24 h was as as that at and PR with ER to Estrogen the transfected PR the effect of E2 by with ER binding to ERE, interference assay by The plasmid two between the and the of transcription. The ER binding to will the of the in the of the gene It has been that of up to into the expression of activity in the of ER Katzenellenbogen B.S. Mol. Cell. Biol. 1992; 12: PubMed Scopus Google Scholar, 1998; PubMed Google Scholar). Cells transfected with were used as transfection controls for treatment. The of treatment in was as the percentage of expression by to that by expression by in cells is on the of that by suggesting interference of activity by cellular MCF-7 and cells, E2 treatment for and h caused a in activity with interference activity was abolished in PR-transfected cells at transfected PR inhibited ER binding to This effect as as h after treatment. The of PR on Cell a of have shown that the effect of E2 on cell was demonstrated after h of treatment expression that the antiestrogenic effect of transfected PR following a of were to this also in cell the effect of E2 on cell was stimulatory in PR-transfected cells up to the of h after treatment. The of cells to of the controls only after h of treatment of MCF-7 and cells were that in control cells the E2 treatment growth after Studies of both gene expression and cell that E2 normally the h of treatment depending on the the PR transfection the metabolism of E2 such that E2 is this a of E2 be growth stimulatory This was by cells in culture over a of and the culture medium was by medium with E2 after and that E2 was to induce an increase of after 24 h in culture in PR-transfected cells. it after h in The stimulatory and following the of E2. This is in to the effect of E2 on vector-transfected cells cells in which the of cells was in cells that of control cells the the that E2 in PR-transfected cells may be metabolized at a that in control cells, and E2 may be to a growth in PR-transfected MCF-7 cells. of E2 in PR-transfected MCF-7 of [3H]E2 in the of and cells were by of in the of following HPLC The whole were that of both a and were present in the The were by acid to interference in [3H]E2 was as it was the only in and its also that of E2 is a of [3H]E2 and this was present in was a decrease of the [3H]E2 and increase of the with in both cell However, in culture medium of cells with that of cells. h of of [3H]E2 was metabolized by cells with by cells. 6 h of of [3H]E2 was metabolized by cells with by cells. It is remarkable that the percentage of [3H]E2 metabolized by cells in 6 h is similar to that by cells in 24 h suggesting a of E2 in PR-transfected cells the vector-transfected percentage of in and for the and E2 control cell cell in a new The of [3H]E2 in is between and This the for and 16α-hydroxyestrone are of the of E2, are insufficient for to the of the in of E2 and E2 in a new also the E2 metabolism is caused by enhanced cellular that after h of with cellular [3H]E2 in and cells was that in control cells MCF-7 and It that enhanced cellular uptake of E2 in PR-transfected cells is one of the for increased metabolism of E2. analysis and of steroid receptors revealed that the steroid receptor to is the ER, followed by the progesterone receptor Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar, E. D. 2003; Scopus Google Scholar). expression of PR in target may from an of The of PR estrogen may to of cells. This study that the expression of PR prior to hampered the function of inhibited E2-mediated gene expression and abolished its growth stimulatory the antiestrogenic effects of have been (14Kraus W.L. Weis K.E. Katzenellenbogen B.S. Mol. Cell. Biol. 1995; 15: 1847-1857Crossref PubMed Scopus (175) Google Scholar, 15Katzenellenbogen B.S. J. Soc. Gynecol. Investig. 2000; 1: S33-S37Crossref Google Scholar, C. Gynecol. Endocrinol. 2001; 15: PubMed Scopus Google Scholar), the antiestrogenic effects of are progesterone antiprogestins such as or were to the antiestrogenic of PR-transfected MCF-7 cells of that the antiestrogenic effect of may be the metabolism of E2. The was that the of E2 on gene expression and cell was only impaired following or h of treatment. analysis revealed no in the ER levels between PR-transfected cells and control cells, that this antiestrogenic effect may be caused by the of E2. the of PR-transfected cells to controls at the E2 was to the growth stimulatory effect in cells. of HPLC analysis of [3H]E2 in the the of enhanced metabolism of [3H]E2 in PR-transfected cells metabolized [3H]E2 in 6 h with a similar percentage metabolized by cells in 24 The study also suggests that increased cellular uptake of E2 is one of the for the increased E2 metabolism in PR-transfected cells. This is conceivable as the metabolism of E2 in culture the cells J. A. 2003; PubMed Scopus Google Scholar). the of 6α-hydroxyestradiol, estriol, and 16α-hydroxyestrone that of the the of the in the to be It has been that and are the main of metabolism L. C. A. R. PubMed Scopus Google Scholar). On the other hand, in MCF-7 cells estrone, and as the main of E2 Biol. PubMed Scopus Google Scholar, Gu J.A. S. A.S. Y. Acad. Sci. 2001; PubMed Scopus Google Scholar, J.A. T. S. P. J.D. Cancer Res. 2001; Google Scholar, N.E. W. Mol. Cell. Endocrinol. 1989; PubMed Scopus Google Scholar). were in our study on their Biol. PubMed Scopus Google Scholar, 2001; 22: PubMed Scopus Google Scholar). The may be because of in experimental conditions such as the and the of in E2, the of E2 used, and the of The between the antiestrogenic effects on gene expression and on cell also the of metabolism of E2 in PR-transfected cells. The effect of E2 the has the that were to cells to 2 of cell the antiestrogenic effect on cell was only observed following h of treatment. E2 of gene expression is a and the effect can be observed at h following E2 E2 was metabolized in PR-transfected cells between 6 and 24 h following E2 a in gene of cell be after of E2 as is shown in It is that the of PR-transfected cells at h is It to that E2 has been to growth that can the effect of E2. such as and have been shown to inhibit the growth of breast cancer cells, effects were only exhibited at high Sutherland R.L. Cancer Res. Google Scholar, T. Y. H. R. L. PubMed Scopus Google Scholar). The E2 of used in this study is to of E2-mediated interference was abolished as as h after treatment in cells. This antiestrogenic to be to be to the of E2. may be to the impaired this the mechanism of for between and ER is It has been Gronemeyer H. Turcotte B. D. Chambon P. Cell. 1989; Full Text PDF PubMed Scopus Google that activity of ER was inhibited in cells by ER and PR in a manner. This inhibition be by the expression level of ER. The study to that PR may with ER for or as to the ER activity. It is also that for binding with such as and that are to the receptor in a for binding T. Chem. 2003; PubMed Scopus Google Scholar). It is transfected PR increased cellular uptake of E2. that the binding of E2 for PR is of progesterone C.L. H.L. J. Endocrinol. PubMed Scopus Google Scholar). a of for progesterone the for E2 be in the of It is for E2 at to to PR However, observed that E2 an inhibition of the PR progesterone response binding in the interference and suggesting an effect of E2 on this is binding remains to be the antiestrogenic potential of has been in an in vivo Breast cancer cells into estrogen-dependent tumors in PR-negative cells, and this is independent of the PR C.A. T. Horwitz K.B. Breast Cancer Res. 2003; PubMed Scopus Google Scholar). The effect was PR as tumors were only the of However, was no of one PR isoform the other in our and PR-B, the two cell have similar potential to the growth stimulatory effect of E2. Studies of gene expression of pS2 and GREB1 that the antiestrogenic potential of is that in This may be to the level of PR in cells of the expression of PR the study demonstrated that estrogen-independent expression of PR in MCF-7 cells the stimulatory effect of E2 on cell and on gene expression in a PR ligand-independent and with ER binding to This antiestrogenic effect of is associated with an metabolism of E2 in PR-transfected MCF-7 cells, which is increased cellular uptake of E2. of by transfected PR may the interaction in a interference The findings open up a new window for a hitherto unknown functional relationship between the PR and ER. This antiestrogenic mechanism is also of therapeutic to the of breast which to antiestrogen and to the tumors that and Chambon from the of and and for the expression vectors hPR1 and and S. of at for the interference

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