Estrogen induces estrogen-related receptor alpha gene expression and chromatin structural changes in estrogen receptor (ER)-positive and ER-negative breast cancer cells

Estrogen-related receptor alpha (ERRalpha), a member of the nuclear receptor superfamily, is closely related to the estrogen receptors (ERalpha and ERbeta). The ERRalpha gene is estrogen-responsive in several mouse tissues and cell lines, and a multiple hormone-response element (MHRE) in the promoter is an important regulatory region for estrogen-induced ERRalpha gene expression. ERRalpha was recently shown to be a negative prognostic factor for breast cancer survival, with its expression being highest in cancer cells lacking functional ERalpha. The contribution of ERRalpha in breast cancer progression remains unknown but may have important clinical implications. In this study, we investigated ERRalpha gene expression and chromatin structural changes under the influence of 17beta-estradiol in both ER-positive MCF-7 and ER-negative SKBR3 breast cancer cells. We mapped the nucleosome positions of the ERRalpha promoter around the MHRE region and found that the MHRE resides within a single nucleosome. Local chromatin structure of the MHRE exhibited increased restriction enzyme hypersensitivity and enhanced histone H3 and H4 acetylation upon estrogen treatment. Interestingly, estrogen-induced chromatin structural changes could be repressed by estrogen antagonist ICI 182 780 in MCF-7 cells yet were enhanced in SKBR3 cells. We demonstrated, using chromatin immunoprecipitation assays, that 17beta-estradiol induces ERRalpha gene expression in MCF-7 cells through active recruitment of co-activators and release of co-repressors when ERRalpha and AP1 bind and ERalpha is tethered to the MHRE. We also found that this estrogen effect requires the MAPK signaling pathway in both cell lines.     

Selective cytotoxicity of ascochlorin in ER-negative human breast cancer cell lines

While agents targeting estrogen receptors are most effective in adjuvant therapy for human breast cancers expressing estrogen receptors after surgery, breast cancers lacking estrogen receptor are clinically serious, because they are highly malignant and exhibit resistance to the usual anti-cancer drugs, including estrogen receptor-antagonists and DNA breaking agents. Here, we found that MX-1, a human breast cancer cell line lacking estrogen receptors, exhibited higher AP-1 activity and expressed higher levels of c-Jun, c-Fos, and Fra-1 when compared with conventional estrogen receptor-positive human breast cancer cell lines. The prenylphenol antibiotic ascochlorin suppressed the AP-1 activity of MX-1 cells, and selectively killed MX-1 cells, partly due to induction of apoptosis. Our results suggest that AP-1 is an effective clinical target molecule for the treatment of estrogen receptor-negative human breast cancer.     

Autologous dendritic cell vaccine for estrogen receptor (ER)/progestin receptor (PR) double-negative breast cancer

Purpose

A wealth of preclinical information, as well as a modest amount of clinical information, indicates that dendritic cell vaccines have therapeutic potential. The aim of this work was to assess the immune response, disease progression, and post-treatment survival of ER/PR double-negative stage II/IIIA breast cancer patients vaccinated with autologous dendritic cells pulsed with autologous tumor lysates.

Methods

Dendritic cell (DC) vaccines were generated from CD14+ precursors pulsed with autologous tumor lysates. DCs were matured with defined factors that induced surface marker and cytokine production. Individuals were immunized intradermally four times. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and lymphocyte subsets were determined for the evaluation of the therapeutic efficiency. Overall survival and disease progression rates were analyzed using Kaplan–Meier curves and compared with those of contemporaneous patients who were not administered DC vaccines.

Results

There were no unanticipated or serious adverse effects. DC vaccines elicited Th1 cytokine secretion and increased NK cells, CD8+ IFN-γ+ cells but decreased the percentage of CD3+ T cells and CD3+ HLA-DR+ T cells in the peripheral blood. Approximately 58% (18/31) of patients had a DTH-positive reaction. There was no difference in overall survival between the patients with and without DC vaccine. The 3-year progression-free survival was significantly prolonged: 76.9% versus 31.0% (with vs. without DC vaccine, p?Conclusion Our findings strongly suggest that tumor lysate-pulsed DCs provide a standardized and widely applicable source of breast cancer antigens that are very effective in evoking anti-breast cancer immune responses.     

Point mutation of estrogen receptor (ER) in the ligand-binding domain changes the pharmacology of antiestrogens in ER-negative breast cancer cells stably expressing complementary DNAs for ER.

The antiestrogen tamoxifen is used in the treatment of hormone-responsive breast cancer. However, therapeutic failure has frequently been observed in both patients and animal models after long term treatment. We have studied the effect of a point mutation that leads to the substitution of Val for Gly at codon 400 in the ligand-binding domain of the estrogen receptor (ER) on estrogenic and antiestrogenic activities of 4-hydroxytamoxifen (4-OHT) and its derivatives. Stable ER transfectants derived from MDA-MB-231 CL10A, an ER-negative breast cancer cell line, have been used in these studies. 4-OHT and its fixed ring derivatives showed more estrogen-like activity in ER transfectants than in MCF-7, an ER-positive breast cancer cell line. In this study, 4-OHT was a partial agonist of cell growth in the transfectant S30 cells, which express the wild-type ER. However, it was a full agonist in the mutant ER transfectant ML alpha 2H, which expressed ER with Val at codon 400. The increased estrogenic activity of 4-OHT in ML alpha 2H cells was not due to the preferential isomerization of trans 4-OHT to cis 4-OHT, since the nonisomerizable fixed ring trans 4-OHT was a partial agonist for cell growth in S30 cells and was a full agonist in ML alpha 2H cells. Transient transfection using a reporter plasmid containing an estrogen response element demonstrated that fixed ring trans 4-OHT had estrogenic activity in ML alpha 2H cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel estrogen receptor (ER) modulators containing various hydrophobic bent-core structures

We previously discovered m-carborane-containing estrogen receptor (ER) modulator 4, which exhibits weak ER-agonistic and antagonistic activities in transactivation assays. With the aim of developing novel ER partial agonists, we designed and synthesized various analogues of 4 with a bent-core structure, that is, pseudo cyclic structure (5), tetrahydropyrimidinone (6), m-benzene (7), adamantane (8), and 9,10-dimethyl-m-carborane (9), in place of the m-carborane moiety. Compound 9 showed greater binding affinity than 4 in ER-binding assay using [6,7-³H]-17β-estradiol and was a more effective partial agonist than 4 in MCF-7 cell proliferation assay. It appears to be a promising candidate as a selective ER modulator (SERM).     

Introduction of estrogen-responsiveness into mammalian cell lines.

We introduced estrogen responsiveness as a new characteristic into rat hepatoma, mouse Ltk- and human HeLatk-cells by transfecting the human estrogen receptor (ER) cDNA. To measure the estrogen response we used Xenopus vitellogenin gene A2 constructs linked to the bacterial CAT gene. Transient cotransfections of the ER cDNA and the vitellogenin gene-CAT constructs containing the estrogen responsive element (ERE) lead to a hormone dependent induction of CAT activity whereas cotransfected vitellogenin gene constructs lacking the ERE are not inducible. Stable transfections of ER cDNA into Ltk- cells give rise to cell clones that are estrogen responsive as shown by transfection of various vitellogenin gene-CAT constructs. These results prove that the transfected ER is biologically active and is sufficient to make a cell estrogen responsive.     

Cloning of the novel isoform of the estrogen receptor beta cDNA (ERbeta isoform M cDNA) from the human testicular cDNA library

Our recent report has revealed the existence of the progesterone receptor (PR) isoform S, which consists of the novel PR exon S and exons 4-8 of the PR gene in the human testicular cDNA library. More recently, we have cloned the human estrogen receptor alpha (ERalpha) isoform S cDNA from the library. The ERalpha isoform S cDNA also contains the novel ERalpha exon S and exons 4-8 of the ERalpha cDNA. Based on these findings, we assumed that the novel isoform of cDNA like the PR- and ERalpha isoforms might exist in the human ER beta (ERbeta). In order to investigate this possibility, we have screened the human testicular cDNA library using the exons 4-8 corresponding sequence of the human ERbeta cDNA. Consequently, we have cloned a novel isoform of the ERbeta cDNA that consists of a previously unidentified 5'-sequence and the exons 5-8 of the ERbeta gene. We termed this isoform cDNA the "ERbeta isoform M cDNA". The 5'-sequence of the ERbeta isoform M cDNA was confirmed to be derived from a novel exon (termed the "exon M") by analysis of the genomic DNA. Moreover, we have analyzed the molecular size of the ERbeta isoform M encoded by the ERbeta isoform M mRNA by transient expression of the ERbeta isoform M cDNA in the 293T cell. The approximately 28 kDa protein, which was recognized by the anti-rat ERbeta antibody against the carboxyl-terminal region, was synthesized in the cells. Thus, we concluded that the ATG in the exon M could be used as the translation initiation codon. This report revealed for the first time the existence of the ERbeta mRNA isoform that is not caused by the skipping of one or more exons, by the alternative usage of the multiple exon 8s, nor by the alternative utilization of the untranslated 5'-exons located on the upstream region of the exon 1.     

ER和PRmRNAs在内异症子宫内膜表达的变化

目的 :探讨雌激素受体 (ER)和孕激素受体 (PR)在子宫内膜异位症 (内异症 )子宫内膜的表达。方法 :利用大鼠内异症动物模型 ,采用逆转录聚合酶链反应 (RT PCR)技术 ,检测子宫内膜ER和PRmRNAs的表达情况。结果 :内异症模型组大鼠异位内膜ER、PRmRNAs的表达低于在位内膜和对照组正常子宫内膜 ,与后两者比较差异有显著性意义 (P <0 .0 1) ;而模型组在位内膜ER、PRmRNAs的表达与正常对照组比较差异无显著性意义 (P >0 .0 5 )。内异症模型组异位内膜ER/PRmRNA比值大于在位内膜和正常子宫内膜ER/PRmRNA比值 (P <0 .0 1)。结论 :内异症大鼠异位内膜ERmRNA表达的相对增高在内异症的发生与发展中起着一定的作用。     

Generation of full-length cDNA recombinant vectors for the transient expression of human fibronectin in mammalian cell lines

In order to study the roles of the different alternatively spliced variants of human fibronectin (FN) as well as of its binding sites and structural domains in the processes of extracellular matrix assembly, cell adhesion, and cell migration, we constructed expression vectors coding for different human full-length FN polypeptides and deleted versions of these constructs. We expressed them transiently in mammalian cells by calcium phosphate transfection and microinjection techniques. While the deleted recombinants were expressed by both transfection and microinjection, the full-length recombinants could be only expressed by microinjection. Calcium phosphate transfection leads to the accumulation of recombinant FN in cytoplasmic vesicles of both matrix-forming and nonforming cells. On the contrary, microinjection leads to the accumulation of recombinant FN in cytoplasmic vesicles in cells that do not form a matrix, but to the rapid incorporation into the extracellular matrix of matrix-forming cells in addition to a cytoplasmic localization. Identical results were obtained when the FN signal and propeptides were replaced by those of E-cadherin.