Cyclin D1 expression is dependent on estrogen receptor function in tamoxifen-resistant breast cancer cells

The development of resistance to tamoxifen, the most common antiestrogen used in the treatment of breast cancer, is a frequent and severe clinical problem. Tamoxifen-resistant tumors are still capable of responding to other hormonal therapies such as those that downregulate estrogen receptor expression. Mechanisms leading to acquisition of tamoxifen-resistant but hormone-sensitive growth are not completely understood. In tamoxifen-sensitive breast cancer cells, tamoxifen inhibits, whereas estrogen induces, expression of cyclin D1, a key cell cycle regulatory protein. Ectopic expression of cyclin D1 can lead to antiestrogen resistance. Thus, to determine whether cyclin D1 is involved in the growth of tamoxifen-resistant cells, we developed several tamoxifen-resistant variants from MCF-7 cells. These variants grow in the absence of estrogen or in the presence of tamoxifen, but their growth is inhibited by estrogen receptor downregulators. We show here that cyclin D1 expression is maintained at comparable levels in all tamoxifen-resistant variants, whereas pS2, another estrogen-regulated protein, is not. The addition of physiological levels of estrogen further stimulates cyclin D1 expression and proliferation. In contrast, treatment with estrogen receptor downregulators decreases cyclin D1 expression and proliferation. Thus, changes in cyclin D1 expression upon second-line hormonal therapy may predict hormonal sensitivity of tamoxifen-resistant tumors. These studies suggest that estrogen receptor mediates cyclin D1 expression and growth of tamoxifen-resistant tumors.     

Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human-breast cancer cells

An estrogen-regulated 52-kDa glycoprotein secreted by MCF7 breast cancer cells was first purified from serum-free conditioned medium by concanavalin-A--Sepharose (ConA--Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52-kDa protein [Garcia et al. (1985) Cancer Res. 45, 709-716]. Using ConA--Sepharose and monoclonal antibody affinity chromatographies, the secreted 52-kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and one single N-terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two-step procedure, applied to MCF7 cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250-fold with a yield of 30%. These components were further separated by high-performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver-stained SDS-PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52-kDa protein and the cellular 34-kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34-kDa protein is given. Western immunoblotting, pulse chase studies and post-translational studies indicate that the 52-kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.     

Changes in oestrogen receptor-alpha and -beta during progression to acquired resistance to tamoxifen and fulvestrant (Faslodex, ICI 182,780) in MCF7 human breast cancer cells

Cell culture models of antioestrogen resistance often involve applying selective pressures of oestrogen deprivation simultaneously with addition of tamoxifen or fulvestrant (Faslodex, ICI 182,780) which makes it difficult to distinguish events in development of antioestrogen resistance from those in loss of response to oestrogen or other components. We describe here time courses of loss of antioestrogen response using either oestrogen-maintained or oestrogen-deprived MCF7 cells in which the only alteration to the culture medium was addition of 10(-6) M tamoxifen or 10(-7) M fulvestrant. In both oestrogen-maintained and oestrogen-deprived models, loss of growth response to tamoxifen was not associated with loss of response to fulvestrant. However, loss of growth response to fulvestrant was associated in both models with concomitant loss of growth response to tamoxifen. Measurement of oestrogen receptor alpha (ERalpha) and oestrogen receptor beta (ERbeta) mRNA by real-time RT-PCR together with ERalpha and ERbeta protein by Western immunoblotting revealed substantial changes to ERalpha levels but very little alteration to ERbeta levels following development of antioestrogen resistance. In oestrogen-maintained cells, tamoxifen resistance was associated with raised levels of ERalpha mRNA/protein. However by contrast, in oestrogen-deprived MCF7 cells, where oestrogen deprivation alone had already resulted in increased levels of ERalpha mRNA/protein, long-term tamoxifen exposure now reduced ERalpha levels. Whilst long-term exposure to fulvestrant reduced ERalpha mRNA/protein levels in the oestrogen-maintained cells to a level barely detectable by Western immunoblotting and non-functional in inducing gene expression (ERE-LUC reporter or pS2), in oestrogen-deprived cells the reduction was much less substantial and these cells retained an oestrogen-induction of both the ERE-LUC reporter gene and the endogenous pS2 gene which could still be inhibited by antioestrogen. This demonstrates that whilst ERalpha can be abrogated by fulvestrant and increased by tamoxifen in some circumstances, this does not always hold true and mechanisms other than alteration to ER must be involved in the development of antioestrogen resistant growth.     

Effects of tamoxifen and 4-hydroxytamoxifen on the pNR-1 and pNR-2 estrogen-regulated RNAs in human breast cancer cells

The estrogenic and antiestrogenic activities of tamoxifen and 4-hydroxytamoxifen have been measured on the expression of two estrogen-regulated RNAs (pNR-1 and pNR-2) in the MCF7 human breast cancer cell line cultured in phenol red-free medium. The two antiestrogens increased the level of the pNR-1 RNA to about 80% of the estradiol-induced level, and the induction by estradiol was not significantly antagonized by either antiestrogen. In contrast, the pNR-2 mRNA was only increased to about 10% of the estradiol-induced level, and its induction by estradiol was antagonized by both tamoxifen and 4-hydroxytamoxifen. Thus, the two RNAs respond in dramatically different ways to these antiestrogens. 4-Hydroxytamoxifen and estradiol have similar affinities for the estrogen receptor; however, the induction of both RNAs by 4-hydroxytamoxifen required a 10-fold higher concentration than estradiol for maximum agonist activity, and a 500-fold molar excess was required to antagonize the induction by estradiol. Tamoxifen has a 20-100-fold lower affinity than estradiol for the estrogen receptor. A 200-fold higher concentration was required for maximum agonist activity and a 10,000-fold molar excess to antagonize the induction by estradiol. These results emphasize the complexity of antiestrogen action in human breast cancer cells.     

Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium

Antiestrogens are efficient inhibitors of estrogen-mediated growth of human breast cancer. Besides inhibiting estradiol-stimulated growth, antiestrogens may have a direct growth-inhibitory effect on estrogen receptor (ER) positive cells and thus be more efficient than aromatase inhibitors, which will only abrogate estrogen-dependent tumor growth. To address this issue, we have used the human breast cancer cell line MCF-7/S9 as a model system which is maintained in a chemically defined medium without serum and estrogen. The addition of estradiol results in an increase in cell growth rate. Thus, the MCF-7/S9 cell line is estrogen-responsive but not estrogen-dependent. Three different types of antiestrogens, namely tamoxifen, ICI 182,780 and EM-652 were found to exert a significant and dose-dependent inhibition of basal growth of MCF-7/S9 cells. The growth-inhibitory effect of the three antiestrogens was prevented by simultaneous estradiol treatment. Antiestrogen treatment also reduced the basal pS2 mRNA expression level, thus indicating spontaneous estrogenic activity in the cells. However, treatment with the aromatase inhibitor had no effect on basal cell growth, excluding that endogenous estrogen synthesis is involved in basal growth. These data demonstrate that in addition to their estrogen antagonistic effect, antiestrogens have a direct growth-inhibitory effect which is ER-mediated. Consequently, in the subset of ER positive breast cancer patients with estrogen-independent tumor growth, antiestrogen therapy may be superior to treatment with aromatase inhibitors which only inhibit estrogen formation but do not affect cancer cell growth in the absence of estrogens.     

Effect of tamoxifen and tamoxifen derivatives on the conversion of estrone-sulfate to estradiol in the R-27 cells, a tamoxifen-resistant line derived from MCF-7 human breast cancer cells

R-27 cells, a tamoxifen-resistant clone of MCF-7 mammary cancer cells, were used to study the effect of tamoxifen and its derivatives (4-hydroxytamoxifen, N-desmethyltamoxifen and cis-tamoxifen) on the conversion of estrone sulfate to estradiol. The present data indicate that (1) tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen and cis-tamoxifen inhibit the uptake of the radioactivity after incubation of these triphenylethylene derivatives with [3H]-estrone sulfate; (2) there is a significant decrease of the conversion of estrone sulfate to estradiol by these antiestrogens; (3) the concentrations of estradiol (cytosol + 0.6 M KCl nuclear extract) which are 293 +/- 50 pg/mg DNA in the control studies (estrone sulfate alone), diminish to 26 +/- 5 pg/mg DNA after addition of tamoxifen, to 9 +/- 2 with 4-hydroxytamoxifen, to 24 +/- 7 with N-desmethyltamoxifen and to 32 +/- 6 with cis-tamoxifen. It is concluded that estrone sulfate can play an important role in the biological responses to estrogens in this breast cancer cell line and tamoxifen and its derivatives block the conversion of estrone sulfate to estradiol. The decrease in concentration of estradiol could be explained by the presence of the estrogen receptor system but other ways of the action of antiestrogens remain to be explored.     

LRP16对乳腺癌MCF-7细胞增殖的影响

用Northern印迹方法检测雌二醇 (17β E2 )对LRP16mRNA表达的时间及剂量依赖性调控作用 .构建LRP16基因启动子序列调控的萤光素酶报告子 (pS0 ) ,并与雌激素受体α和 β(ERα和ERβ)表达载体共转染COS 7和MCF 7细胞后测定萤光素酶活性 .将LRP16基因的表达载体转染MCF 7细胞 ,测定过表达LRP16对细胞的生长特性的影响 .17β E2 使MCF 7细胞中LRP16mRNA表达水平增加 ,增加幅度未显示出 17β E2 培养时间和剂量的依赖性 .pS0 与ERα表达载体共转染细胞的相对萤光素酶活性较非共转染组 (对照组 )及pS0 ERβ表载体共转染组升高 5～ 10倍 .LRP16基因过表达促进MCF 7细胞的增殖 .研究表明 ,雌激素可能通过ERα上调乳腺癌MCF 7细胞LRP16基因的表达并促进细胞增殖     

Identification and characterization of estrogen-regulated RNAs in human breast cancer cells

Two cDNA libraries have been constructed with RNA prepared from the estrogen-responsive breast cancer cell lines, MCF7 and ZR 75. They were screened by differential hybridization for estrogen-regulated sequences. A total of 11 different RNAs were isolated from the MCF7 cell cDNA library and four from the ZR 75 cell cDNA library. Only two sequences were isolated from both libraries. The levels of the 13 different RNAs are induced between 2.5- and 100-fold by estrogen in MCF7 cells. The expression and regulation by estrogen of the RNAs was examined in eight different human tumor cell lines. The relative abundance of each RNA varied in the different cell lines. The expression of three RNAs (pNR-1, pNR-2, and pNR-25) was detected only in estrogen-responsive breast cancer cells. The sequences that were expressed in all eight cell lines were regulated by estrogen only in the three estrogen-responsive breast cancer cell lines. The response of the RNAs to other classes of steroids and to different concentrations of estrogen was characterized in more detail. The extent to which different concentrations of estradiol induced each RNA varied, but half-maximal induction of most of the RNAs occurred between 2 and 5 X 10(-11) M. The time at which increased RNA levels were first detected following exposure to estradiol also varied. Estrogen increased the levels of some RNAs within 15 min, while for others there was a lag of 4 h.     

Induction of estrogen-regulated genes differs in immortal and tumorigenic human mammary epithelial cells expressing a recombinant estrogen receptor.

Studies on estrogen receptor (ER)-positive human breast cancer cell lines have shown that estrogen treatment positively modulates the expression of the genes encoding transforming growth factor-alpha (TGF alpha), 52-kDa cathepsin-D, and pS2. To determine whether these genes would be similarly regulated by estrogens in normal human mammary epithelial cells, we stably transfected immortal nontumorigenic human mammary epithelial cells with an ER-encoding expression vector. ER-negative tumor cells were also transfected for comparison. Levels of TGF alpha and 52-kDa cathepsin-D mRNA were enhanced by estrogen treatment of both ER-transfected immortal and tumorigenic cells, demonstrating that the ER by itself is sufficient to elicit estrogenic regulation of the expression of these genes. In contrast, expression of the pS2 gene was detected only in the ER-transfected tumor cells. The ER in both cell lines is capable of recognizing the pS2 promoter, however, since estrogen enhanced the activity of an introduced pS2-CAT reporter plasmid in transient expression analyses. These and other experiments with somatic cell hybrids between the immortal cells and ER+/pS2+ MCF-7 tumor cells, where pS2 gene expression is extinguished, support the conclusion that the immortal nontumorigenic cells encode gene products that block endogenous pS2 expression. These results also imply that such repressors are not active in the tumor cells.     

Anti-microRNA-222 (anti-miR-222) and -181B suppress growth of tamoxifen-resistant xenografts in mouse by targeting TIMP3 protein and modulating mitogenic signal

We have shown earlier that miR-221 and -222 are up-regulated in tamoxifen-resistant MCF-7 (OHT(R)) cells and Her2-positive human breast tumors when compared with Her2 negative tumors. In this study, we report markedly enhanced expression of miR-181b in OHT(R) cells and endocrine-resistant tumors. Further, anti-miR-222 or -181b in combination with tamoxifen suppressed growth of tamoxifen-resistant xenografts in mice. Luciferase reporter assay and expression analysis showed that TIMP3, a tissue metalloproteinase inhibitor, is a common target of miR-221/222 and -181b. In situ hybridization and immunohistochemical analysis demonstrated reciprocal relationships between TIMP3 and miR-221/222/181b expression in primary human breast carcinomas. Ectopic expression of TIMP3 inhibited growth of the OHT(R) cells, and its depletion in MCF-7 cells reduced sensitivity to tamoxifen in vitro and in vivo. EGF-induced MAPK and AKT phosphorylation were significantly higher in OHT(R) cells and miR-221/222-overexpressing MCF-7 cells than in control cells, which suggests modulation of mitogenic signaling by TIMP3 and the miRs. On the contrary, phosphoMAPK and phosphoAKT levels were diminished in TIMP3-overexpressing OHT(R) cells and increased in TIMP3-depleted MCF-7 cells. Low levels of estrogen or tamoxifen elicited similar differences in phosphoMAPK levels in these cells. Reduced levels of TIMP3 facilitated growth of tamoxifen-resistant cells by alleviating its inhibitory effect on ADAM10 and ADAM17, which are critical for OHT(R) cell growth. In conclusion, miR-221/222 and -181b facilitate growth factor signaling in tamoxifen-resistant breast cancer by down-regulating TIMP3, and corresponding anti-miRs can be used to render these tumors responsive to tamoxifen.     

Comparative study of the short-term effects of a novel selective estrogen receptor modulator, ospemifene, and raloxifene and tamoxifen on rat uterus

To investigate the differential short-term effects of selective estrogen receptor (ER) modulators (SERMs) on uterus, we treated adult ovariectomized rats with a novel SERM, ospemifene (Osp), two previously established SERMs (tamoxifen and raloxifene (Ral)) and estradiol. The expression of two estrogen-regulated early response genes c-fos and vascular endothelial growth factor (VEGF), and DNA synthesis were analysed at 1-24 h after treatment of ovariectomized rats. Induction of c-fos mRNA by each of the SERMs showed a biphasic pattern with peaks at 3 and 20 h, respectively. The maximum level of VEGF mRNA was observed at 1 h after raloxifene and 6 h after tamoxifen or ospemifene treatment. Maximum levels of the c-fos and VEGF mRNA after raloxifene treatment were higher than those seen after treatments with E2 or a corresponding dose of tamoxifen or ospemifene. DNA synthesis was significantly increased by ospemifene, tamoxifen and raloxifene both in luminal and glandular epithelium. The stimulation was transient, peaking at 16 h. In comparison, the maximum level observed at 16 h after E2 treatment sustained at least until 24 h. DNA synthesis in stromal cells was increased by the SERMs but not by E2 at 24 h. When treated together with E2, the SERMs were able to antagonise E2-stimulated DNA synthesis at 16 h. Our results demonstrate that the initial response of uterus to ospemifene, raloxifene and tamoxifen includes activation of early response genes and even transient stimulation of DNA synthesis in spite of their different long-term effects. However, the early stimulatory events may be mediated by different mechanisms leading to diverging pathways in various tissue compartments and development of differential SERM-specific long-term responses of uterus.