Shuk-Mei Ho

Comparative Studies of the Estrogen Receptors β and α and the Androgen Receptor in Normal Human Prostate Glands, Dysplasia, and in Primary and Metastatic Carcinoma

An antibody, GC-17, thoroughly characterized for its specificity for estrogen receptor-β (ER-β), was used to immunolocalize the receptor in histologically normal prostate, prostatic intraepithelial neoplasia, primary carcinomas, and in metastases to lymph nodes and bone. Comparisons were made between ER-β, estrogen receptor-α (ER-α), and androgen receptor (AR) immunostaining in these tissues. Concurrently, transcript expression of the three steroid hormone receptors was studied by reverse transcriptase-polymerase chain reaction analysis on laser capture-microdissected samples of normal prostatic acini, dysplasias, and carcinomas. In Western blot analyses, GC-17 selectively identified a 63-kd protein expressed in normal and malignant prostatic epithelial cells as well as in normal testicular and prostatic tissues. This protein likely represents a posttranslationally modified form of the long-form ER-β, which has a predicted size of 59 kd based on polypeptide length. In normal prostate, ER-β immunostaining was predominately localized in the nuclei of basal cells and to a lesser extent stromal cells. ER-α staining was only present in stromal cell nuclei. AR immunostaining was variable in basal cells but strongly expressed in nuclei of secretory and stromal cells. Overall, prostatic carcinogenesis was characterized by a loss of ER-β expression at the protein and transcript levels in high-grade dysplasias, its reappearance in grade 3 cancers, and its diminution/absence in grade 4/5 neoplasms. In contrast, AR was strongly expressed in all grades of dysplasia and carcinoma. Because ER-β is thought to function as an inhibitor of prostatic growth, androgen action, presumably mediated by functional AR and unopposed by the β receptor, may have provided a strong stimulus for aberrant cell growth. With the exception of a small subset of dysplasias in the central zone and a few carcinomas, ER-α-stained cells were not found in these lesions. The majority of bone and lymph node metastases contained cells that were immunostained for ER-β. Expression of ER-β in metastases may have been influenced by the local microenvironment in these tissues. In contrast, ER-α-stained cells were absent in bone metastases and rare in lymph nodes metastases. Irrespective of the site, AR-positive cells were found in all metastases. Based on our recent finding of anti-estrogen/ER-β-mediated growth inhibition of prostate cancer cells in vitro, the presence of ER-β in metastatic cells may have important implications for the treatment of late-stage disease.

Relation of DNA Methylation of 5′-CpG Island of ACSL3 to Transplacental Exposure to Airborne Polycyclic Aromatic Hydrocarbons and Childhood Asthma

In a longitudinal cohort of ∼700 children in New York City, the prevalence of asthma (>25%) is among the highest in the US. This high risk may in part be caused by transplacental exposure to traffic-related polycyclic aromatic hydrocarbons (PAHs) but biomarkers informative of PAH-asthma relationships is lacking. We here hypothesized that epigenetic marks associated with transplacental PAH exposure and/or childhood asthma risk could be identified in fetal tissues. Mothers completed personal prenatal air monitoring for PAH exposure determination. Methylation sensitive restriction fingerprinting was used to analyze umbilical cord white blood cell (UCWBC) DNA of 20 cohort children. Over 30 DNA sequences were identified whose methylation status was dependent on the level of maternal PAH exposure. Six sequences were found to be homologous to known genes having one or more 5′-CpG island(s) (5′-CGI). Of these, acyl-CoA synthetase long-chain family member 3 (ACSL3) exhibited the highest concordance between the extent of methylation of its 5′-CGI in UCWBCs and the level of gene expression in matched fetal placental tissues in the initial 20 cohort children. ACSL3 was therefore chosen for further investigation in a larger sample of 56 cohort children. Methylation of the ACSL3 5′-CGI was found to be significantly associated with maternal airborne PAH exposure exceeding 2.41 ng/m3 (OR = 13.8; p<0.001; sensitivity = 75%; specificity = 82%) and with a parental report of asthma symptoms in children prior to age 5 (OR = 3.9; p<0.05). Thus, if validated, methylated ACSL3 5′CGI in UCWBC DNA may be a surrogate endpoint for transplacental PAH exposure and/or a potential biomarker for environmentally-related asthma. This exploratory report provides a new blueprint for the discovery of epigenetic biomarkers relevant to other exposure assessments and/or investigations of exposure-disease relationships in birth cohorts. The results support the emerging theory of early origins of later life disease development.

Female reproductive disorders: the roles of endocrine-disrupting compounds and developmental timing

OBJECTIVE To evaluate the possible role of endocrine-disrupting compounds (EDCs) on female reproductive disorders emphasizing developmental plasticity and the complexity of endocrine-dependent ontogeny of reproductive organs. Declining conception rates and the high incidence of female reproductive disruptions warrant evaluation of the impact of EDCs on female reproductive health. DESIGN Publications related to the contribution of EDCs to disorders of the ovary (aneuploidy, polycystic ovary syndrome, and altered cyclicity), uterus (endometriosis, uterine fibroids, fetal growth restriction, and pregnancy loss), breast (breast cancer, reduced duration of lactation), and pubertal timing were identified, reviewed, and summarized at a workshop. CONCLUSION(S) The data reviewed illustrate that EDCs contribute to numerous human female reproductive disorders and emphasize the sensitivity of early life-stage exposures. Many research gaps are identified that limit full understanding of the contribution of EDCs to female reproductive problems. Moreover, there is an urgent need to reduce the incidence of these reproductive disorders, which can be addressed by correlative studies on early life exposure and adult reproductive dysfunction together with tools to assess the specific exposures and methods to block their effects. This review of the EDC literature as it relates to female health provides an important platform on which womens health can be improved.

Estrogen receptor (ER)-β isoforms: A key to understanding ER-β signaling

Estrogen receptor beta (ER-β) regulates diverse physiological functions in the human body. Current studies are confined to ER-β1, and the functional roles of isoforms 2, 4, and 5 remain unclear. Full-length ER-β4 and -β5 isoforms were obtained from a prostate cell line, and they exhibit differential expression in a wide variety of human tissues/cell lines. Through molecular modeling, we established that only ER-β1 has a full-length helix 11 and a helix 12 that assumes an agonist-directed position. In ER-β2, the shortened C terminus results in a disoriented helix 12 and marked shrinkage in the coactivator binding cleft. ER-β4 and -β5 completely lack helix 12. We further demonstrated that ER-β1 is the only fully functional isoform, whereas ER-β2, -β4, and -β5 do not form homodimers and have no innate activities of their own. However, the isoforms can heterodimerize with ER-β1 and enhance its transactivation in a ligand-dependent manner. ER-β1 tends to form heterodimers with other isoforms under the stimulation of estrogens but not phytoestrogens. Collectively, these data support the premise that (i) ER-β1 is the obligatory partner of an ER-β dimer, whereas the other isoforms function as variable dimer partners with enhancer activity, and (ii) a single functional helix 12 in a dimer is sufficient for gene transactivation. Thus, ER-β behaves like a noncanonical type-I receptor, and its action may depend on differential amounts of ER-β1 homo- and heterodimers formed upon stimulation by a specific ligand. Our findings have provided previously unrecognized directions for studying ER-β signaling and design of ER-β-based therapies.

Environmental Epigenetics and Asthma: Current Concepts and Call for Studies

Recent studies suggest that epigenetic regulation (heritable changes in gene expression that occur in the absence of alterations in DNA sequences) may in part mediate the complex gene-by-environment interactions that can lead to asthma. The variable natural history of asthma (i.e., incidence and remission of symptoms) may be a result of epigenetic changes, such as DNA methylation, covalent histone modifications, microRNA changes, and chromatin alterations, after early or later environmental exposures. Findings from multiple epidemiologic and experimental studies indicate that asthma risk may be modified by epigenetic regulation. One study suggested that the transmission of asthma risk may occur across multiple generations. Experimental studies provide substantial in vitro data indicating that DNA methylation of genes critical to T-helper cell differentiation may induce polarization toward or away from an allergic phenotype. Despite this initial progress, fundamental questions remain that need to be addressed by well-designed research studies. Data generated from controlled experiments using in vivo models and/or clinical specimens collected after environmental exposure monitoring are limited. Importantly, cohort-driven epigenetic research has the potential to address key questions, such as those concerning the influence of timing of exposure, dose of exposure, diet, and ethnicity on susceptibility to asthma development. There is immense promise that the study of environmental epigenetics will help us understand a theoretically preventable environmental disease.

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: The case of Bisphenol A

Background In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. Objectives We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. Discussion Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. Conclusions Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Estrogens and anti-estrogens: Key mediators of prostate carcinogenesis and new therapeutic candidates

Despite the historical use of estrogens in the treatment of prostate cancer (PCa) little is known about their direct biological effects on the prostate, their role in carcinogenesis, and what mechanisms mediate their therapeutic effects on PCa. It is now known that estrogens alone, or in synergism with an androgen, are potent inducers of aberrant growth and neoplastic transformation in the prostate. The mechanisms of estrogen carcinogenicity could be mediated via induction of unscheduled cell proliferation or through metabolic activation of estrogens to genotoxic metabolites. Age‐related changes and race‐/ethnic‐based differences in circulating or locally formed estrogens may explain differential PCa risk among different populations. Loss of expression of estrogen receptor (ER)‐β expression during prostate carcinogenesis and prevention of estrogen‐mediated oxidative damage could be exploited in future PCa prevention strategies. Re‐expression of ER‐β in metastatic PCa cells raises the possibility of using ER‐β‐specific ligands in triggering cell death in these malignant cells. A variety of new estrogenic/anti‐estrogenic/selective estrogen receptor modulator (SERM)‐like compounds, including 2‐methoxyestradiol, genistein, resveratrol, licochalcone, Raloxifene, ICI 182,780, and estramustine are being evaluated for their potential in the next generation of PCa therapies. Increasing numbers of patients self‐medicate with herbal formulations such as PC‐SPES. Some of these compounds are selective ER‐β ligands, while most of them have minimal interaction with ER‐α. Although many may inhibit testosterone production by blockade of the hypothalamal–pituitary–testis axis, the most effective agents also exhibit direct cytostatic, cytotoxic, or apoptotic action on PCa cells. Some of them are potent in interfering with tubulin polymerization, blocking angiogenesis and cell motility, suppressing DNA synthesis, and inhibiting specific kinase activities. Further discovery of other compounds with potent apoptotic activities but minimal estrogen action should promote development of a new generation of effective PCa preventive or treatment regimens with few or no side‐effects due to estrogenicity. Further advancement of our knowledge of the role of estrogens in prostate carcinogenesis through metabolic activation of estrogens and/or ER‐mediated pathways will certainly result in better preventive or therapeutic modalities for PCa.

Dynamic Regulation of Estrogen Receptor-β Expression by DNA Methylation During Prostate Cancer Development and Metastasis

Estrogen receptor (ER)-beta is thought to exert anti-proliferative effects in the normal prostate but supports prostate cancer (PCa) cell survival. We previously reported that the receptors expression declined as PCa developed in the gland but reappeared in lymph node and bone metastases. To investigate whether hypermethylation was the underlying mechanism for these phenomena, we first identified two CpG islands (CGIs) encompassing 41 CpG dinucleotides, located separately in the untranslated exon 0N and the promoter region of ER-beta. Using immunostained, laser capture-microdissected samples from 56 clinical specimens, we demonstrated an inverse relationship exists between the extent of ER-beta CGI methylation and receptor expression in normal, hyperplastic, premalignant, and malignant foci of the prostate and in lymph node and bone metastases. Treatment of PCa cell lines (LNCaP and DU145), that express little ER-beta mRNA, with a demethylating agent increased levels of receptor expression thus corroborating our in vivo findings that methylation is involved in ER-beta silencing. Methylation centers in the promoter region and exon 0N were identified by hierarchical cluster analysis of bisulfite sequencing data obtained from 710 alleles. Methylation at these centers was insignificant in normal epithelium, reached 80 to 90% in grade 4/5 PCa, but declined to less than 20% in bone metastases. In addition, progressive methylation spreading from the exonic CGI to the promoter CGI, which correlated with loss of ER-beta expression, was detected in microdissected samples and in cell cultures. Using a new class of methylated oligonucleotides that mediate sequence-specific methylation in cellulo, we demonstrated that methylation of the promoter CGI, but not the exonic CGIs, led to transcriptional inactivation of ER-beta. Our results present the first evidence that epigenetic regulation of ER-beta is a reversible and tumor stage-specific process and that gene silencing via methylated oligonucleotides may have therapeutic potential in the treatment of advanced PCa.

Epigenetic reprogramming and imprinting in origins of disease

The traditional view that gene and environment interactions control disease susceptibility can now be expanded to include epigenetic reprogramming as a key determinant of origins of human disease. Currently, epigenetics is defined as heritable changes in gene expression that do not alter DNA sequence but are mitotically and transgenerationally inheritable. Epigenetic reprogramming is the process by which an organism’s genotype interacts with the environment to produce its phenotype and provides a framework for explaining individual variations and the uniqueness of cells, tissues, or organs despite identical genetic information. The main epigenetic mediators are histone modification, DNA methylation, and non-coding RNAs. They regulate crucial cellular functions such as genome stability, X-chromosome inactivation, gene imprinting, and reprogramming of non-imprinting genes, and work on developmental plasticity such that exposures to endogenous or exogenous factors during critical periods permanently alter the structure or function of specific organ systems. Developmental epigenetics is believed to establish “adaptive” phenotypes to meet the demands of the later-life environment. Resulting phenotypes that match predicted later-life demands will promote health, while a high degree of mismatch will impede adaptability to later-life challenges and elevate disease risk. The rapid introduction of synthetic chemicals, medical interventions, environmental pollutants, and lifestyle choices, may result in conflict with the programmed adaptive changes made during early development, and explain the alarming increases in some diseases. The recent identification of a significant number of epigenetically regulated genes in various model systems has prepared the field to take on the challenge of characterizing distinct epigenomes related to various diseases. Improvements in human health could then be redirected from curative care to personalized, preventive medicine based, in part, on epigenetic markings etched in the “margins” of one’s genetic make-up.

Environmental epigenetics of asthma: An update

Asthma, a chronic inflammatory disorder of the airway, is influenced by interplay between genetic and environmental factors now known to be mediated by epigenetics. Aberrant DNA methylation, altered histone modifications, specific microRNA expression, and other chromatin alterations orchestrate a complex early-life reprogramming of immune T-cell response, dendritic cell function, macrophage activation, and a breach of airway epithelial barrier that dictates asthma risk and severity in later life. Adult-onset asthma is under analogous regulation. The sharp increase in asthma prevalence over the past 2 or 3 decades and the large variations among populations of similar racial/ethnic background but different environmental exposures favors a strong contribution of environmental factors. This review addresses the fundamental question of whether environmental influences on asthma risk, severity, and steroid resistance are partly due to differential epigenetic modulations. Current knowledge on the epigenetic effects of tobacco smoke, microbial allergens, oxidants, airborne particulate matter, diesel exhaust particles, polycyclic aromatic hydrocarbons, dietary methyl donors and other nutritional factors, and dust mites is discussed. Exciting findings have been generated by rapid technological advances and well-designed experimental and population studies. The discovery and validation of epigenetic biomarkers linked to exposure, asthma, or both might lead to better epigenotyping of risk, prognosis, treatment prediction, and development of novel therapies.