Oestrogen regulates oestrogen receptor mRNA levels in an oestrogen-responsive human breast cancer cell line

The EFM-19 human breast cancer cell line contains high levels of oestrogen receptor mRNA and is oestrogen responsive for growth. The oestrogen receptor gene appears to be specifically regulated by oestrogens in EFM-19 cells. The induction by oestradiol is half-maximal in the presence of 3 x 10(-11) M oestradiol. LY117018 is a potent antioestrogen with a similar affinity to oestradiol for the oestrogen receptor. LY117018 completely blocks the induction of the oestrogen receptor mRNA by oestradiol when it is present in a 50-fold molar excess. The ability of oestrogens to control the levels of their own receptor in human breast cancer cells has implications for the understanding of oestrogen responsiveness and the amelioration of endocrine therapy.     

Oestrogen regulates cathepsin D mRNA levels in oestrogen responsive human breast cancer cells.

A cDNA library was constructed from the mRNA of the ZR-75 oestrogen responsive human breast cancer cell line and screened for oestrogen regulated mRNA sequences. Of the recombinants isolated, 30 contained cDNA that corresponded to a single mRNA species of 2.1 kb that was induced between 10 and 15 fold by oestradiol treatment. The sequence of the entire open reading frame and 3' non-coding region of the mRNA was determined and shown to encode the aspartyl protease cathepsin D. The induction of cathepsin D mRNA is specific for oestrogen and is maximal at 3 X 10(-10) M. Cathepsin D mRNA levels were increased by oestrogen in 3 oestrogen responsive breast cancer cell lines. Cathepsin D mRNA was expressed but not regulated in an oestrogen receptor negative breast tumour cell line, BT 20, and in 2 other malignant cell lines, Hela and A431.     

Effects of oestrogen and the antioestrogens, tamoxifen and LY117018, on four oestrogen-regulated RNAs in the EFM-19 breast cancer cell line

The EFM-19 cell line is a new breast cancer cell line whose proliferation has been reported to be stimulated by oestrogens and inhibited by the antioestrogen tamoxifen. Oestrogen receptor mRNA levels are higher in EFM-19 cells than in other oestrogen-responsive cell lines. The levels of four oestrogen-inducible RNAs [pNR-1, pNR-2, pNR-25 and pNR-100] were measured in EFM-19 cells. Oestradiol treatment increased the levels of the four regulated RNAs between 3-fold (pNR-100) and greater than 100-fold (pNR-2). The induction was half maximal between 1.5 x 10(-11) and 1.5 x 10(-10) M oestradiol. The effects of two antioestrogens, tamoxifen and LY117018, were measured on the expression of the oestrogen-regulated RNAs. Tamoxifen was a partial oestrogen agonist for the induction of the pNR-1 and pNR-25 RNAs but had very little effect on the pNR-2 and pNR-100 RNA levels. The pNR-2 RNA levels were less induced by tamoxifen in EFM-19 cells than in MCF-7 cells. LY117018 did not increase the levels of any RNA. The oestrogen-induced levels of the four RNAs were reduced by both antioestrogens to the RNA levels present in cells treated with the antioestrogens alone. LY117018 was at least 100-fold more potent than tamoxifen as an oestrogen antagonist.     

Effects of the antioestrogen, ICI 164,384, on oestrogen induced RNAs in MCF-7 cells

The effects of ICI 164,384 on the expression of six oestrogen-regulated RNAs (pNR-1, pNR-2, pNR-13, pNR-17, pNR-25 and pNR-100) and the 46 kDa secreted protein were measured in MCF-7 cells. In marked contrast to tamoxifen, an antioestrogen commonly used in the treatment of breast cancer, ICI 164,384 administered alone had little or no effect on the RNAs or protein. ICI 164,384 completely inhibited the induction of the RNAs and 46 kDa protein by oestradiol. Although ICI 164,384 has an affinity for the human oestrogen receptor only slightly less than that of oestradiol, half maximal inhibition of oestradiol action was attained with between a 50 and 150-fold molar excess of ICI 164,384. The pNR-1 RNA is induced by tamoxifen but this induction was abolished by ICI 164,384. Thus, ICI 164,384 acts as a potent antioestrogen for the regulation of the expression of specific oestrogen-responsive genes in human breast cancer cells.     

Monohydroxytamoxifen: an antioestrogen with high affinity for the chick oviduct oestrogen receptor

The monohydroxylated derivative of tamoxifen (a non-steroidal triaryl ethylene antioestrogen) shows an apparent affinity (Ki = 0.2 nM) for the chick oviduct oestrogen receptor which is higher than that of oestradiol itself, and ～ 10 times higher than that of tamoxifen. Administered $\text{in}\text{vivo}$ with oestradiol benzoate, it inhibited the increase of tissue growth, progesterone receptor content, ornithine decarboxylase activity (ODC), and ovalbumin and conalbumin synthesis, and also inhibited the oestradiol induced increase of ODC $\text{in}\text{vitro}$. It did not display any oestrogenic effect by itself. We conclude that antioestrogenic action may be exhibited by a molecule with higher affinity binding to the oestrogen receptor than oestradiol itself. Metabolic studies demonstrated that the antioestrogenic action of tamoxifen is not due to its prior conversion to monohydroxytamoxifen.     

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.     

Oestrogen receptor gene structure and function in breast cancer

The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoRI, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance.     

Molecular changes associated with the acquisition of oestrogen hypersensitivity in MCF-7 breast cancer cells on long-term oestrogen deprivation

The growth dependence of many breast cancers on oestrogen has been exploited therapeutically by oestrogen deprivation, but almost all patients eventually develop resistance largely by unknown mechanisms. Wild-type (WT) MCF-7 cells were cultured in oestrogen-deficient medium for 90 weeks in order to establish a long-term oestrogen-deprived MCF-7 (LTED) which eventually became independent of exogenous oestrogen for growth. After 15 weeks of quiescence (LTED-Q), basal growth rate increased in parallel with increasing oestrogen sensitivity. While 10⁻⁹ M oestradiol (E2) maximally stimulated WT growth, the hypersensitive LTED (LTED-H) were maximally growth stimulated by 10⁻¹³ M E2. By week 50, hypersensitivity was apparently lost and the cells became oestrogen independent (LTED-I), although the pure antioestrogen ICI182780 still inhibited cell growth and reversed the inhibitory effect of 10⁻⁹ M E2 at 10⁻¹² to 10⁻⁷ M. Tamoxifen (10⁻⁷ to 10⁻⁶ M) had a partial agonist effect on WT, but had no stimulatory effect on LTED. Whilst LTED cells have a low progesterone receptor (PgR) expression in all phases, oestrogen receptor (ER) a expression was, on average, elevated five- and seven-fold in LTED-H and LTED-I, respectively, and serine118 was phosphorylated. ERβ expression was up-regulated and the levels of insulin receptor substrate 1 (IRS-1) remained low throughout all phases. The levels of RIP140 mRNA appeared to decrease to approximately 50% of the WT message in LTED-Q and remained constant into the hypersensitive phase. No significant changes were observed in the expression of SUG-1, TIF-1 and SMRT in LTED. The overall changes in nuclear receptor interacting proteins do not appear to be involved in the hypersensitivity. Thus, the resistance of these human breast cancer cells to oestrogen-deprivation appears to be due to acquired hypersensitivity which may be explained in part by increased levels of and phosphorylated ER.     

Multihormone regulation of MMTV-LTR in transfected T-47-D human breast cancer cells

Multihormonal regulation on the long terminal repeat (LTR) region of mouse mammary tumour virus (MMTV) has been studied using T-47-D human breast cancer cells stably transfected with the steroid sensitive LTR-C3 chimaeric gene. The specificity of steroid action on transfected LTR sequences has been compared with regulation of endogenous cellular markers. We conclude that the hormone response element of the LTR can be induced by physiological concentrations of androgen, progestin and glucocorticoid. 17 beta-Oestradiol did not regulate the LTR at physiological levels but an effect was found at 10(-6) M. This effect was not inhibited by antioestrogen nor was it reproduced by the synthetic oestrogen diethylstilboestrol suggesting such effects do not occur via the oestrogen receptor. The antioestrogens tamoxifen and transhydroxytamoxifen do not induce the LTR. No significant steroid competition was found in LTR regulation: whilst oestradiol did not act at physiological concentration it did not interfere with induction by androgen, progestin or glucocorticoid. Such gene regulation did not simply follow receptor status of the cells nor was it reflected in patterns of growth regulation by steroids. The implications of these findings on the mechanism of steroid hormone action are discussed.     

Are breast tumours resistant to tamoxifen also resistant to pure antioestrogens?

A substantial proportion of patients with breast cancer are treated with the antioestrogen tamoxifen. As with other endocrine therapies, clinical experience has shown that some tumours in which growth is initially attenuated by tamoxifen treatment become resistant to continued drug treatment and resume growth. The mechanisms underlying the development of tamoxifen resistance have yet to be described but represent an important focus of research with the aim of defining what other therapies might be effective following tamoxifen treatment. Secondly, an understanding of tamoxifen resistance might suggest means to develop more effective agents for primary treatment of the disease. The development of pure antioestrogens, for example ICI 164,384 and ICI 182,780, which differ pharmacologically from tamoxifen in being entirely free of oestrogen partial-agonist activity, together with cell and animal models of tamoxifen resistant human breast cancer, has revealed one mechanism which might be of considerable clinical significance. Pure antioestrogens were shown to inhibit the proliferation of a greater proportion of tumor cells than tamoxifen in vitro, a differential effect that was attributed to the oestrogenic activity of tamoxifen. Subsequently, cell culture studies have shown that breast cancer cell lines selected for resistance to tamoxifen can still remain sensitive to the growth inhibitory action of pure antioestrogens. Similarly, the growth of human breast tumours in nude mice, which is initially attenuated by tamoxifen but then resumes, can be inhibited by pure antioestrogens. Both types of experiment are consistent with the view that tamoxifen resistance in these model systems is due to the oestrogenic action of tamoxifen. Thus, it can be predicted that in some patients whose tumours recur during tamoxifen therapy, a further response to pure antioestrogen treatment might occur. Studies to examine this hypothesis are currently being undertaken with ICI 182,780. One mechanism which might account for the experimental observations is an intrinsic heterogeneity amongst breast tumour cells in their response to tamoxifen, i.e. that there are at least two different populations of cells; one population which responds to tamoxifen as an antioestrogen and one which “reads” tamoxifen as an oestrogen. The growth advantage thus conferred on the latter population would lead to its predominance. If this is what actually happens in a proportion of human tumours, it can be argued that primary treatment of the tumour with a pure antioestrogen, rather than tamoxifen, would be preferred since a more complete and longer-lasting response would be predicted. Recent comparative studies with human breast tumours grown in nude mice support these predictions.     

Induction and inhibition of estradiol hydroxylase activities in MCF-7 human breast cancer cells in culture

The effects of estradiol, progesterone, and tamoxifen on the activity of estradiol 2- and 16 alpha-hydroxylases were studied in human breast cancer cell cultures using a radiometric assay. After 5 days' exposure to these compounds, incubations in the presence of either [2-3H]estradiol or [16 alpha-3H]estradiol as substrate were carried out. In MCF-7 cells, estradiol (10(-8) M), progesterone (10(-6) M) and tamoxifen (10(-6) M) significantly increased 16 alpha-hydroxylase activity (estradiol; 21% progesterone 10% to 32%; tamoxifen 21% to 31%; P less than 0.01). Synergistic effects were observed when the cells were successively exposed to tamoxifen and progesterone. Simultaneous treatment with tamoxifen plus estradiol or estradiol plus progesterone showed no change from estradiol alone. On the other hand, although estradiol had no direct effects on 2-hydroxylase activity, tamoxifen decreased this enzymatic activity significantly at 10(-6) M (23% to 37%). Progesterone acted synergistically to further decrease this reaction. Treatment with only progesterone caused an increase in 2-hydroxylation. In contrast, a subline of MCF-7 cells with low estrogen receptor levels showed only minimal enzyme-hormone responses. Likewise, treatment of the estrogen receptor-negative MDA-MB-231 human breast cancer cell line with these compounds showed no effects on either 2- or 16 alpha-hydroxylase activity. In the progesterone receptor-rich T47D cell line, estradiol decreased both activities while progesterone increased both.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel binding site for a synthetic progestagen in breast cancer cells

A novel, high-affinity saturable binding site for the synthetic 19-nor testosterone progestagen, 17 alpha-ethinyl-13 beta-ethyl 17 beta-hydroxy-4,15-oestradiene-3-one (gestodene) has been detected using a sensitive affinity chromatography technique. This binding site is present in a range of malignant breast-derived cells lines, regardless of the presence of oestrogen and progesterone receptors, but is absent from endometrial carcinoma cells that contain both oestrogen and progesterone receptors. Competition studies show that this binding is not attributable to the receptors for the progestagens, androgens, glucocorticoids or mineralocorticoids. Cytosolic gestodene binding is refractory to competition with oestradiol but nuclear gestodene binding is completely abolished by oestradiol. The binding of oestradiol to the oestrogen receptor is reduced 40-50% by competition with gestodene. Non-dissociating polyacrylamide gel electrophoresis and size-exclusion high performance liquid chromatography reveal that this binding activity is associated with a protein of mean molecular mass 47 +/- 9 kDa. Ligand binding studies with a range of other cell lines indicates that this binding site appears to be specific to breast cancer cells. These data show the presence of a partly oestrogen competable novel binding protein in breast cancer cells which does not appear to be due to any of the conventional steroid receptors.     

Oestradiol and tamoxifen inhibit murine Colon 38 cancer growth and increase the cytotoxic effect of fluorouracil

The poor efficacy of reference chemotherapy (fluorouracil -FU) in colon cancer has resulted in a constant search for agents which could augment the action of FU. Epidemiological data, such as the decreased risk of colorectal cancer among menopausal women receiving hormonal replacement therapy, indicate the role of oestrogen in the pathogenesis of this disease. The differences between normal and neoplastic colon cells in the expression of oestrogen receptor beta (ERbeta) could confirm this association. However, the direct influence of oestrogen or tamoxifen (SERM, selective oestrogen receptor modulator) on colon cancer growth has rarely been studied. The aim of the present study was to examine the direct effects of various concentrations of oestradiol and tamoxifen (10(-4) to 10(-12) M), applied alone or together with FU, on the growth of murine Colon 38 cancer in vitro as assessed by three colorimetric methods: Mosmann's method, incorporation of BrdU into cell nuclei and the TUNEL method. At high concentrations oestradiol and tamoxifen decreased the cancer growth in a dose- and time-dependent manner (the Mosmann and BrdU methods) and at some concentrations augmented the cytotoxic action of FU (Mosmann's method). Tamoxifen exerted a very early and potent inhibitory effect, inducing even total cancer growth inhibition at the concentration of 10(-4) M (the Mosmann and BrdU methods). All the substances studied at different concentrations and at different incubation time points increased the apoptosis of tumour cells (the TUNEL method). The results indicate that oestradiol and tamoxifen inhibit Colon 38 cancer growth and increase the cytotoxic effect of FU, which confirms the role of sex steroids in colon carcinogenesis and even suggests new therapeutic schemes.     

ICI 182,780, a new antioestrogen with clinical potential.

Previous studies in this laboratory identified a series of 7 alpha-alkylamide analogues of 17 beta-oestradiol which are pure antioestrogens. Among this initial lead series of compounds, exemplified by ICI 164,384, none was of sufficient in vivo potency to merit serious consideration as a candidate for clinical evaluation. Further structure-activity studies identified a new compound, ICI 182,780, 7 alpha-[9-(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-1,3,5(10)- triene-3,17 beta-diol, with significantly increased antioestrogenic potency. The antiuterotrophic potency of ICI 182,780 is more than 10-fold greater than that of ICI 164,384. ICI 182,780 has no oestrogen-like trophic activity and, like ICI 164,384 is peripherally selective in its antioestrogenic effects. The increased in vivo potency of ICI 182,780 was also reflected, in part, by intrinsic activity at the oestrogen receptor and in the growth inhibitory potency of ICI 182,780 in MCF-7 human breast cancer cells. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. Sustained antioestrogenic effects of ICI 182,780, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, the antitumour activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in athymic mice where, over a 1 month period, a single injection of ICI 182,780 in oil suspension achieved effects comparable with those of daily tamoxifen treatment. Thus, ICI 182,780 provides the opportunity to evaluate clinically the potential therapeutic benefits of complete blockade of oestrogen effects in endocrine-responsive human breast cancer.     

Oestrogen and progesterone receptors in chick oviduct chromatin after administration of oestradiol, progesterone or anti-oestrogen.

Oestrogen-primed and withdrawn chicks were injected with oestradiol benzoate, progesterone, and/or the anti-oestrogens tamoxifen and 4-hydroxytamoxifen. Oestrogen receptors were studied in oviduct chromatin solubilized by mild digestion of purified nuclei with micrococcal nuclease. After a single injection of oestradiol benzoate, ultracentrifugation on sucrose gradients of chromatin extracts labelled with [3H]-oestradiol showed two peaks of oestradiol binding sites, sedimenting at 13--14 S and 7--8 S. After repeated injections of oestradiol benzoate, the 13--14 S peak increased more than the 7--8 S peak. After injection of anti-oestrogen alone or together with oestradiol benzoate, no [3H]oestradiol-binding or 4-hydroxy[3H]tamoxifen-binding peaks were detected in the chromatin. Injection of progesterone also produced an increase of the 13--14 S and 7--8 S chromatin oestradiol receptor. Progesterone receptor could only by detected in chromatin early after progesterone administration, and it sedimented in density gradients with the 12 S mononucleosome fraction. Tamoxifen injected together with progesterone gave higher levels of 13--14 S oestrogen binding sites than did progesterone alone. The presence of a 13--14 S peak of oestrogen binding sites in hormonal situations which promote a biological response in the chick oviduct, and the absence of this peak after administration of anti-oestrogens, suggest that this subfraction of chromatin contains elements involved in gene regulation.     

Expression of progesterone receptor isoforms A and B is differentially regulated by estrogen in different breast cancer cell lines

Progesterone action in target tissues is mediated through two progesterone receptor (PR) isoforms, PR-A and PR-B, which display different regulatory functions in target cells. Relative expression ratio of these isoforms varies depending on cell and tissue types. Here, we studied the regulation of PR isoform expression by estradiol (E(2)), insulin, IGF-1 and cAMP in different breast cancer cell lines. Although, E(2) induced PR expression in all cell lines studied, the expression ratio of PR-A/PR-B induced by E(2) was dependent on the cell line. The differential regulation of the isoforms was also seen at the mRNA level suggesting that the PR-A and PR-B promoters are differentially regulated by E(2) in different breast cancer cells. Insulin, IGF-1 or cAMP previously reported to induce PR expression however failed to alter the PR expression in our study. This is the first report describing that in different breast cancer cell lines the expression of PR-A and PR-B is regulated by E(2) in a distinct way.     

Bcl-2, survivin and variant CD44 v7-v10 are downregulated and p53 is upregulated in breast cancer cells by progesterone: Inhibition of cell growth and induction of apoptosis

Progesterone inhibits the proliferation of normal breast epithelial cells in vivo, as well as breast cancer cells in vitro. But the biologic mechanism of this inhibition remains to be determined. We explored the possibility that an antiproliferative activity of progesterone in breast cancer cell lines is due to its ability to induce apoptosis. Since p53, bcl-2 and survivin genetically control the apoptotic process, we investigated whether or not these genes could be involved in the progesterone-induced apoptosis.We found a maximal 90% inhibition of cell proliferation with T47-D breast cancer cells after exposure to 10 M progesterone for 72 h. Control progesterone receptor negative MDA-231 cancer cells were unresponsive to 10 M progesterone. The earliest sign of apoptosis is translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane and can be monitored by the calcium-dependent binding of annexin V in conjunction with flow cytometry. After 24 h of exposure to 10 M progesterone, cytofluorometric analysis of T47-D breast cancer cells indicated 43% were annexin V-positive and had undergone apoptosis and no cells showed signs of cellular necrosis (propidium iodide negative). After 72 h of exposure to 10 M progesterone, 48% of the cells had undergone apoptosis and 40% were annexin V positive/propidium iodide positive indicating signs of necrosis. Control untreated cancer cells did not undergo apoptosis. Evidence proving apoptosis was also demonstrated by fragmentation of nuclear DNA into multiples of oligonucleosomal fragments.After 24 h of exposure of T47-D cells to either 1 or 10 M progesterone, we observed a marked down-regulation of protooncogene bcl-2 protein and mRNA levels. mRNA levels of survivin and the metastatic variant CD44 v7-v10 were also downregulated. Progesterone increased p53 mRNA levels.These results demonstrate that progesterone at relative high physiological concentrations, but comparable to those seen in plasma during the third trimester of human pregnancy, exhibited a strong antiproliferative effect on breast cancer cells and induced apoptosis.     

Elevated CRB3 expression suppresses breast cancer stemness by inhibiting β‐catenin signalling to restore tamoxifen sensitivity

Tamoxifen is a first‐line drug for hormone therapy (HT) in oestrogen receptor‐positive breast cancer patients. However, 20% to 30% of those patients are resistant to tamoxifen treatment. Cancer stem cells (CSCs) have been implicated as one of the mechanisms responsible for tamoxifen resistance. Our previous study indicated that decreased expression of the CRB3 gene confers stem cell characteristics to breast cancer cells. In the current investigation, we found that most of the breast cancer patient tissues resistant to tamoxifen were negative for CRB3 protein and positive for β‐catenin protein, in contrast to their matched primary tumours by immunohistochemical analysis. Furthermore, expression of CRB3 mRNA and protein was low, while expression of β‐catenin mRNA and protein was high in tamoxifen resistance cells (LCC2 and T47D TamR) contrast to their corresponding cell lines MCF7 and T47D. Similarly, CRB3 overexpression markedly restored the tamoxifen sensitivity of TamR cells by the MTT viability assay. Finally, we found that CRB3 suppressed the stemness of TamR cells by inhibiting β‐catenin signalling, which may in turn lead to a decrease in the breast cancer cell population. Furthermore, these findings indicate that CRB3 is an important regulator for breast cancer stemness, which is associated with tamoxifen resistance.     

Progesterone therapy results in partial reversibility of uterine abnormalities of the adult anovulatory rat.

The effects of progesterone therapy (5 mg, administered subcutaneously daily for 6 days) on the abnormal uterus of adult anovulatory Wistar rats have been studied. These rats, rendered anovulatory by neonatal treatment with testosterone propionate or clomiphene citrate, displayed severe hyperplasia and metaplasia of the uterine luminal epithelium and a disproportionately high content of nuclear oestrogen receptor, as a result of constant oestrogen stimulation unrelieved by progesterone [White, Moore, Elder & Lim (1981) Biochem. J. 196, 557-565]. Progesterone therapy resulted in the virtual elimination of the hyperplasia and metaplasia and a corresponding decrease in the content of nuclear oestrogen receptor with the proportion of the unoccupied nuclear receptor being increased to values exhibited by normal cyclic females. There was also a decrease in the content of progestin receptors, a putative index of oestrogenic stimulation. Further, in the testosterone-treated group, progesterone therapy resulted in the restoration of oestrogen receptor translocational responses to oestradiol stimulation. Progesterone treatment of these anovulatory rats thus provides a model system for investigating the biochemical mechanisms underlying progestin antagonism and regulation of oestrogen-stimulated cell proliferation.     

Seasonal changes in oestrogen receptor affinity in the domestic rabbit, Oryctolagus cuniculus

Over the course of 1 year the Ka value of uterine oestrogen receptor for oestradiol varied 10-fold from a low of 0.259 +/- 0.065 X 10(9) M-1 in early spring to 2.21 +/- 0.21 X 10(9) M-1 in fall. There were no significant changes in receptor number. In addition, administration of oestradiol or progesterone to ovariectomized rabbits resulted in significant reductions in measureable oestrogen receptor affinity. Although these variations in oestrogen receptor Ka values parallel reproductive success in the domestic rabbit, no causal relationship has been established.