David J. Shapiro

Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation

We have demonstrated, by DNA excess filter hybridizations to pulse-labeled cell RNA, that estrogen selectively stabilizes Xenopus liver vitellogenin mRNA against cytoplasmic degradation. The half-life of vitellogenin mRNA is approximately 3 weeks in the presence of estrogen and 16 hr after estrogen is withdrawn from the culture medium. Total poly(A) mRNA exhibits the same half-life (16 hr) in the presence or absence of estrogen. The rapid cytoplasmic degradation of vitellogenin mRNA in the absence of estrogen is fully reversible upon restimulation with estrogen, indicating that nuclear modification of vitellogenin RNA transcripts is not responsible for their stability. Intermediate levels of vitellogenin mRNA stability and changes in the relative rate of vitellogenin gene transcription are not observed late in estrogen induction, when vitellogenin mRNA levels plateau. Instead, Xenopus liver cells achieve fine control over the level of vitellogenin mRNA through down-regulation of the overall rate of total nuclear RNA synthesis.

Activation of the p38 Mitogen-activated Protein Kinase Pathway by Estrogen or by 4-Hydroxytamoxifen Is Coupled to Estrogen Receptor-induced Apoptosis

17β-Estradiol (E2) or the antiestrogen, 4-hydroxytamoxifen (OHT), induce apoptosis in stably transfected estrogen receptor (ER)-positive HeLa-ER5 cells. p38 mitogen-activated protein kinase is implicated in cellular processes involving apoptosis. The p38 kinase inhibitor, SB203580, partially protects HeLa-ER5 cells against apoptosis induced by E2 or by OHT. E2 induces the p38 pathway 12–36-fold in ER-positive cell lines, while OHT induces p38 activity 2–5-fold. In an ER-positive cell line selected for resistance to E2-induced apoptosis, E2 no longer induced p38, and the ER no longer bound to the estrogen response element, while OHT induced both p38 and apoptosis. In cells selected for resistance to OHT-induced apoptosis, OHT no longer induced p38, while E2 induced p38 and apoptosis, and transactivated an estrogen response element-containing reporter gene. In MCF-7 cells, whose growth is stimulated by estrogen, E2 did not induce p38 or apoptosis, while OHT induced both p38 and apoptosis, and SB203580 protected against OHT-induced apoptosis. This work shows that E2 and OHT activate the p38 pathway, suggests that they use different pathways for p38 activation, and links activation of the p38 pathway to apoptosis induced by E2 and by OHT.

Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending.

We have used circular permutation analysis to determine whether binding of purified Xenopus laevis estrogen receptor DNA-binding domain (DBD) to a DNA fragment containing an estrogen response element (ERE) causes the DNA to bend. Gel mobility shift assays showed that DBD-DNA complexes formed with fragments containing more centrally located EREs migrated more slowly than complexes formed with fragments containing EREs near the ends of the DNA. DNA bending standards were used to determine that the degree of bending induced by binding of the DBD to an ERE was approximately 34 degrees. A 1.55-fold increase in the degree of bending was observed when two EREs were present in the DNA fragment. These in vitro studies suggest that interaction of nuclear receptors with their hormone response elements in vivo may result in an altered DNA conformation.

The immune system and inflammation in breast cancer.

During different stages of tumor development the immune system can either identify and destroy tumors, or promote their growth. Therapies targeting the immune system have emerged as a promising treatment modality for breast cancer, and immunotherapeutic strategies are being examined in preclinical and clinical models. However, our understanding of the complex interplay between cells of the immune system and breast cancer cells is incomplete. In this article, we review recent findings showing how the immune system plays dual host-protective and tumor-promoting roles in breast cancer initiation and progression. We then discuss estrogen receptor α (ERα)-dependent and ERα-independent mechanisms that shield breast cancers from immunosurveillance and enable breast cancer cells to evade immune cell induced apoptosis and produce an immunosuppressive tumor microenvironment. Finally, we discuss protumorigenic inflammation that is induced during tumor progression and therapy, and how inflammation promotes more aggressive phenotypes in ERα positive breast cancers.

In Vitro Genetic Analysis of the RNA Binding Site of Vigilin, a Multi-KH-Domain Protein

ABSTRACT The function(s) and RNA binding properties of vigilin, a ubiquitous protein with 14 KH domains, remain largely obscure. We recently showed that vigilin is the estrogen-inducible protein in polysome extracts which binds specifically to a segment of the 3′ untranslated region (UTR) of estrogen-stabilized vitellogenin mRNA. In order to identify consensus mRNA sequences and structures important in binding of vigilin to RNA, before vigilin was purified, we developed a modified in vitro genetic selection protocol. We subsequently validated our selection procedure, which employed crude polysome extracts, by testing natural and in vitro-selected RNAs with purified recombinant vigilin. Most of the selected up-binding mutants exhibited hypermutation of G residues leading to a largely unstructured, single-stranded region containing multiple conserved (A) n CU and UC(A) n motifs. All eight of the selected down-binding mutants contained a mutation in the sequence (A) n CU. Deletion analysis indicated that approximately 75 nucleotides are required for maximal binding. Using this information, we predicted and subsequently identified a strong vigilin binding site near the 3′ end of human dystrophin mRNA. RNA sequences from the 3′ UTRs of transferrin receptor and estrogen receptor, which lack strong homology to the selected sequences, did not bind vigilin. These studies describe an aproach to identifying long RNA binding sites and describe sequence and structural requirements for interaction of vigilin with RNAs.

Regulation of pathways of mRNA destabilization and stabilization

The level of an mRNA in the cytoplasm represents a balance between the rate at which the mRNA precursor is synthesized in the nucleus and the rates of nuclear RNA processing and export and cytoplasmic mRNA degradation. Although most studies of gene expression have focused on gene transcription and in the area of eukaryotic mRNA degradation, but to provide a short general discussion of the importance of mRNA degradation and its regulation and a brief overview of recent findings and present knowledge. The overview is followed by a more in-depth discussion of one of the several pathways for mRNA degradation. We concentrate on the pathway for regulated mRNA degradation mediated by mRNA-binding proteins and endonucleases that cleave within the body of mRNAs. As a potential example of this type of control, we focus on the regulated degradation of the egg yolk precursor protein vitellogenin on the mRNA-binding protein vigilin and the mRNA endonuclease polysomal ribonuclease 1 (PMR-1).

Interplay between the levels of estrogen and estrogen receptor controls the level of the granzyme inhibitor, proteinase inhibitor 9 and susceptibility to immune surveillance by natural killer cells

Estrogens promote cell proliferation and metastases in several human cancers. Here, we describe a different action of estrogens likely to contribute to tumor development-blocking immunosurveillance. In breast cancer cells, increasing concentrations of estrogen induce increasing levels of the granzyme B inhibitor, SerpinB9/proteinase inhibitor 9 (PI-9) and progressively block cell death induced by NK92 natural killer (NK) cells, but do not block killing by a second NK cell line, NKL cells. RNA interference knockdown of PI-9 abolishes estrogens ability to block NK92 cell-induced cytotoxicity. Expressing elevated levels of estrogen receptor α (ERα) increases the induced level of PI-9, and makes tamoxifen (TAM), but not raloxifene or ICI 182,780, a potent inducer of PI-9. At elevated levels of ERα, induction of PI-9 by estradiol or TAM blocks killing by both NK92 and NKL cells. When the Erk pathway is activated with epidermal growth factor, the concentration of estrogen required to induce a protective level of PI-9 is reduced to 10 pM. Elevated concentrations of estrogen and ER may provide a dual selective advantage to breast cancer cells by controlling PI-9 levels and thereby blocking immunosurveillance. Expressing elevated levels of ERα reveals a potentially important difference in the effects of TAM, raloxifene and ICI 182,780 on immunosurveillance in breast cancer.

Estrogen regulation of gene transcription and mRNA stability

This chapter discusses that to study the molecular basis for steroid hormone regulation of gene expression, model systems are employed based on the estrogen control of liver cell differentiation and the induction of the liver messenger RNAs (mRNAs) coding for the Xenopus laevis egg yolk precursor protein, vitellogenin, for the serum retinol binding protein and for the Xenopus estrogen receptor. The experimental system initially employed in studies is the estrogen induction of the vitellogenin mRNAs in liver cells of the amphibian, X. laevis. The chapter reviews that rationale for a detailed study of the regulatory mechanisms used to achieve the induction of vitellogenin mRNA is derived from both in vivo studies and experiments using primary liver explant cultures. In the experiments explained in the chapter, quantitative RNA dot hybridization is used to measure the kinetics of vitellogenin mRNA induction. The data demonstrates that estrogen induces vitellogenin mRNA from undetectable levels to approximately 50,000 molecules per cell, at which time it comprises approximately half of the cells mRNA.

A New Small Molecule Inhibitor of Estrogen Receptor α Binding to Estrogen Response Elements Blocks Estrogen-dependent Growth of Cancer Cells

Estrogen receptor α (ERα) plays an important role in several human cancers. Most current ERα antagonists bind in the receptor ligand binding pocket and compete for binding with estrogenic ligands. Instead of the traditional approach of targeting estrogen binding to ER, we describe a strategy using a high throughput fluorescence anisotropy microplate assay to identify small molecule inhibitors of ERα binding to consensus estrogen response element (cERE) DNA. We identified small molecule inhibitors of ERα binding to the fluorescein-labeled (fl)cERE and evaluated their specificity, potency, and efficacy. One small molecule, theophylline, 8-[(benzylthio)methyl]-(7CI,8CI) (TPBM), inhibited ERα binding to the flcERE (IC50 ∼ 3 μm) and inhibited ERα-mediated transcription of a stably transfected ERE-containing reporter gene. Inhibition by TPBM was ER-specific, because progesterone and glucocorticoid receptor transcriptional activity were not significantly inhibited. In tamoxifen-resistant breast cancer cells that overexpress ERα, TPBM inhibited 17β-estradiol (E2)-ERα (IC50 9 μm) and 4-hydroxytamoxifen-ERα-mediated gene expression. Chromatin immunoprecipitation showed TPBM reduced E2·ERα recruitment to an endogenous estrogen-responsive gene. TPBM inhibited E2-dependent growth of ERα-positive cancer cells (IC50 of 5 μm). TPBM is not toxic to cells and does not affect estrogen-independent cell growth. TPBM acts outside of the ER ligand binding pocket, does not act by chelating the zinc in ER zinc fingers, and differs from known ERα inhibitors. Using a simple high throughput screen for inhibitors of ERα binding to the cERE, a small molecule inhibitor has been identified that selectively inhibits ERα-mediated gene expression and estrogen-dependent growth of cancer cells.

RUNX3 acts as a tumor suppressor in breast cancer by targeting estrogen receptor α

Transcription factor RUNX3 is inactivated in a number of malignancies, including breast cancer, and is suggested to function as a tumor suppressor. How RUNX3 functions as a tumor suppressor in breast cancer remains undefined. Here, we show that about 20% of female Runx3+/− mice spontaneously developed ductal carcinoma at an average age of 14.5 months. Additionally, RUNX3 inhibits the estrogen-dependent proliferation and transformation potential of ERα-positive MCF-7 breast cancer cells in liquid culture and in soft agar and suppresses the tumorigenicity of MCF-7 cells in severe combined immunodeficiency mice. Furthermore, RUNX3 inhibits ERα-dependent transactivation by reducing the stability of ERα. Consistent with its ability to regulate the levels of ERα, expression of RUNX3 inversely correlates with the expression of ERα in breast cancer cell lines, human breast cancer tissues and Runx3+/− mouse mammary tumors. By destabilizing ERα, RUNX3 acts as a novel tumor suppressor in breast cancer.