Yuet-Kin Leung

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.

Estrogen receptor β2 and β5 are associated with poor prognosis in prostate cancer, and promote cancer cell migration and invasion

Estrogens play a pivotal role in the development and progression of prostate cancer (PCa). Their actions are mediated by estrogen receptors (ERs), particularly ERβ in the prostate epithelium. With the discovery of ERβ isoforms, data from previous studies that focused principally on the wild-type ERβ (ERβ1) may not be adequate in explaining the still controversial role of ERβ(s) in prostate carcinogenesis. In this study, using newly generated isoform-specific antibodies, immunohistochemistry (IHC) was performed on a tumor microarray comprised of 144 specimens. IHC results were correlated with pathological and clinical follow-up data to delineate the distinct roles of ERβ1, ERβ2, and ERβ5 in PCa. ERβ2 was commonly found in the cytoplasm and was the most abundant isoform followed by ERβ1 localized predominantly in the nucleus, and ERβ5 was primarily located in the cytoplasm. Logistic regression analyses demonstrated that nuclear ERβ2 (nERβ2) is an independent prognostic marker for prostate specific antigen (PSA) failure and postoperative metastasis (POM). In a Kaplan–Meier analysis, the combined expression of both nERβ2 and cytoplasmic ERβ5 identified a group of patients with the shortest POM-free survival. Cox proportional hazard models revealed that nERβ2 predicted shorter time to POM. In concordance with IHC data, stable, ectopic expression of ERβ2 or ERβ5 enhanced PCa cell invasiveness but only PCa cells expressing ERβ5 exhibited augmented cell migration. This is the first study to uncover a metastasis-promoting role of ERβ2 and ERβ5 in PCa, and show that the two isoforms, singularly and conjointly, have prognostic values for PCa progression. These findings may aid future clinical management of PCa.

Environmental Epigenetics and Its Implication on Disease Risk and Health Outcomes

This review focuses on how environmental factors through epigenetics modify disease risk and health outcomes. Major epigenetic events, such as histone modifications, DNA methylation, and microRNA expression, are described. The function of dose, duration, composition, and window of exposure in remodeling the individuals epigenetic terrain and disease susceptibility are addressed. The ideas of lifelong editing of early-life epigenetic memories, transgenerational effects through germline transmission, and the potential role of hydroxylmethylation of cytosine in developmental reprogramming are discussed. Finally, the epigenetic effects of several major classes of environmental factors are reviewed in the context of pathogenesis of disease. These include endocrine disruptors, tobacco smoke, polycyclic aromatic hydrocarbons, infectious pathogens, particulate matter, diesel exhaust particles, dust mites, fungi, heavy metals, and other indoor and outdoor pollutants. We conclude that the summation of epigenetic modifications induced by multiple environmental exposures, accumulated over time, represented as broad or narrow, acute or chronic, developmental or lifelong, may provide a more precise assessment of risk and consequences. Future investigations may focus on their use as readouts or biomarkers of the totality of past exposure for the prediction of future disease risk and the prescription of effective countermeasures.

Estrogens and Antiestrogens as Etiological Factors and Therapeutics for Prostate Cancer

Abstract:  Mounting evidence supports a key role played by estrogen or estrogen in synergy with an androgen, in the pathogenesis of prostate cancer (PCa). New experimental data suggest that this process could begin as early as prenatal life. During adulthood, estrogen carcinogenicity is believed to be mediated by the combined effects of hormone‐induced, unscheduled cell proliferation and bioactivation of estrogens to genotoxic carcinogens. Increased bioavailability of estrogen through age‐dependent increases in conversion from androgen could also be a contributing factor. Individual variations and race‐/ethnic‐based differences in circulating or locally formed estrogens or in tissue estrogen responsiveness may explain differential PCa risk among individuals or different populations. Estrogen receptor (ER)‐α and ER‐β are the main mediators of estrogen action in the prostate. However, ER‐β is the first ER subtype expressed in the fetal prostate. During cancer development, ER‐β expression is first lost as tumors progress into high grade in the primary site. Yet, its reexpression occurs in all metastatic cases of PCa. A change in cytosine methylation in a regulatory CpG island located in the proximal promoter of ER‐β may constitute an “on/off” switch for reversible regulation of ER‐β expression. A variety of estrogenic/antiestrogenic/selective estrogen receptor modulator (SERM)‐like compounds have been shown to use non‐ERE pathways, such as tethering of ER‐β to NF‐κB binding proteins, Sp2, or Ap1 for gene transactivation. These findings open new avenues for drug design that now focuses on developing a new generation of estrogen‐based PCa therapies with maximal proapoptotic action but few or no side effects.

ENVIRONMENTAL MANGANESE EXPOSURE IN RESIDENTS LIVING NEAR A FERROMANGANESE REFINERY IN SOUTHEAST OHIO: A PILOT STUDY

Manganese (Mn) is an essential element, yet is neurotoxic in excess. The majority of Mn research has been conducted on occupationally exposed adults with few studies focused on an environmentally exposed population. Marietta, OH is home to one of the largest airborne Mn emission sources in the United States, a ferromanganese refinery. In preparation for a community-based participatory research study, a preliminary pilot study was initiated to characterize the communitys exposure to Mn in ambient air and to evaluate the relationship between biological indices of Mn exposure and genes associated with Mn metabolism in Marietta area residents. Participants in the pilot study were recruited through newspaper advertisement, fliers and direct mailing. Exposure to ambient Mn was estimated using an air pollution dispersion model, AERMOD. A total of 141 residents participated in the pilot study ranging in age from 2 to 81 years. Estimated annual average ambient air Mn concentrations in the study area obtained from AERMOD varied from 0.02 to 2.61 microg/m(3). Mean blood and hair Mn values were 9.12 microg/L (SD 3.90) and 5.80 microg/g (SD 6.40 microg/g), respectively and were significantly correlated (r=0.30, p<0.01). Blood and hair Mn was significantly associated within families (r=0.27, p=<0.02 and r=0.43, p<0.01), respectively. The relationship between hair Mn and estimated ambient air Mn became significant when genes for iron metabolism were included in linear models. The preliminary ambient air and biological concentrations of Mn found in this population demonstrate the need for further research into potential health effects.

Herceptin conjugated PLGA-PHis-PEG pH sensitive nanoparticles for targeted and controlled drug delivery

A dual functional nano-scaled drug carrier, comprising of a targeting ligand and pH sensitivity, has been made in order to increase the specificity and efficacy of the drug delivery system. The nanoparticles are made of a tri-block copolymer, poly(d,l lactide-co-glycolide) (PLGA)-b-poly(l-histidine) (PHis)-b-polyethylene glycol (PEG), via nano-precipitation. To provide the nanoparticle feature of endolysosomal escape and pH sensitivity, poly(l-histidine) was chosen as a proton sponge polymer. Herceptin, which specifically binds to HER2 antigen, was conjugated to the nanoparticles through click chemistry. The nanoparticles were characterized via dynamic light scattering (DLS) and transmission electron microscopy (TEM). Both methods showed the sizes of about 100nm with a uniform size distribution. The pH sensitivity was assessed by drug releases and size changes at different pH conditions. As pH decreased from 7.4 to 5.2, the drug release rate accelerated and the size significantly increased. During in vitro tests against human breast cancer cell lines, MCF-7 and SK-BR-3 showed significantly increased uptake for Herceptin-conjugated nanoparticles, as compared to non-targeted nanoparticles. Herceptin-conjugated pH-sensitive nanoparticles showed the highest therapeutic effect, and thus validated the efficacy of a combined approach of pH sensitivity and active targeting.

AP-2 regulates the transcription of estrogen receptor (ER)-beta by acting through a methylation hotspot of the 0N promoter in prostate cancer cells.

We reported previously that the loss of expression of estrogen receptor (ER)-β during the development of prostate cancer (PCa) is associated with methylation of a CpG island located in the 5′-flanking sequence of the 0N promoter. Three methylation hotspots, referred to as centers 1, 2 and 3, were identified in the CpG island. In this study, we demonstrated that a 581-bp region with these three centers within it is sufficient for the promoter activity in PCa cells. Deletion analyses indicated that center 1 (16 bp), with a putative activator protein-2 (AP-2) binding site, is essential for gene transactivation. Chromatin immunoprecipitation assays showed that AP-2α occupies a short sequence containing center 1. Forced expression of AP-2α or -2γ, but not -2β, increased activity of the ERβ 0N promoter and the accumulation of mRNA. Conversely, siRNA-mediated AP-2α and -2γ knockdown reduced levels of ERβ transcript and promoter activity. Quantitative reverse transcription–PCR showed that AP-2α and -2γ are the predominant transcripts expressed in PCa cells, and levels of ERβ transcript correlate with levels of these AP-2 transcripts among different PCa cell lines. These results provide the first evidence that ERβ is an AP-2-regulated gene. They also support the hypothesis that certain cis-acting elements are methylation hotspots susceptible to epigenetic modifications during cancer progression.

Estrogen receptor-beta and breast cancer: translating biology into clinical practice.

Estrogen receptor (ER) β was discovered over a decade ago. The design of most studies on this receptor was based on knowledge of its predecessor, ERα. Although breast cancer (BCa) has been a main focus of ERβ research, its precise roles in breast carcinogenesis remain elusive. Data from in vitro models have not always matched those from observational or clinical studies. Several inherent factors may contribute to these discrepancies: (a) several ERβ spliced variants are expressed at the protein level, and isoform-specific antibodies are unavailable for some variants; (b) post-translational modifications of the receptor regulate receptor functions; (c) the role of the receptor differs significantly depending on the type of ligands, cis-elements, and co-regulators that interact with the receptor; and (d) the diversity of distribution of the receptor among intracellular organelles of BCa cells. This review addresses the gaps in knowledge in ERβ research as it pertains to BCa regarding the following questions: (1) is ERβ a tumor suppressor in BCa?; (2) do ERβ isoforms play differential roles in breast carcinogenesis?; (3) do nuclear signaling and extranuclear ERβ signaling differ in BCa?; (4) what are the consequences of post-translational modifications of ERβ in BCa?; (5) how do co-regulators and interacting proteins increase functional diversity of ERβ?; and (6) how do the types of ligand and regulatory cis-elements affect the action of ERβ in BCa?. Insights gained from these key questions in ERβ research should help in prevention, diagnosis/prognosis, and treatment of BCa.

Forkhead box protein 3 (FOXP3) hypermethylation is associated with diesel exhaust exposure and risk for childhood asthma.

Kelly J. Brunst, Ph.D.1,2,†, Yuet-Kin Leung, Ph.D.2,3, Patrick H. Ryan, Ph.D.1,2,4, Gurjit K. Khurana Hershey, M.D., Ph.D.5, Linda Levin, Ph.D.1,2, Hong Ji, Ph.D.5, Grace K. LeMasters, Ph.D.1, and Shuk-Mei Ho, Ph.D.2,3,6 1Division of Epidemiology and Biostatistics, University of Cincinnati College of Medicine, 3223 Eden Avenue, Kettering Building, Cincinnati, OH 2Center for Environmental Genetics, University of Cincinnati College of Medicine, 3223 Eden Avenue, Kettering Building, Cincinnati, OH 3Division of Environmental Genetics and Molecular Toxicology, Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Kettering Building, Cincinnati, OH 4Division of Biostatistics and Epidemiology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, MLC 7037, Cincinnati, OH 5Division of Asthma Research, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, MLC 7037, Cincinnati, OH 6Cincinnati Veteran Affairs Medical Center, Merit Scholar, 3200 Vine Street, Cincinnati, OH

Overexpression of Cytochrome P450 1A1 and Its Novel Spliced Variant in Ovarian Cancer Cells: Alternative Subcellular Enzyme Compartmentation May Contribute to Carcinogenesis

Epithelial ovarian cancer derived from the human ovarian surface epithelium (HOSE) is the leading cause of death from gynecologic malignancies among American women. Metabolic activation of endogenous and exogenous chemicals by cytochrome P450 (CYP) class I enzymes has been implicated in its etiology. In this study, we showed overexpression of CYP1A1 mRNA, but not CYP1B1 transcripts, in ovarian cancer cell lines when compared with primary cultures or immortalized HOSE cell lines. Importantly, we identified a novel, enzymatically active, spliced variant of CYP1A1 (CYP1A1v) formed by excision of an 84-bp cryptic intron in exon 2. CYP1A1v is overexpressed in ovarian cancer cell lines and exhibits a unique subcellular distribution restricted to the nucleus and mitochondria, contrary to the endoplasmic reticulum localization of the wild-type enzyme. In concordance, total CYP1A1 activity, as measured by the ethoxyresorufin O-deethylase assay, was detected in mitochondrial, nuclear, and microsomal fractions of ovarian cancer cells but was notably absent in all subcellular fractions of HOSE cells. Immunocytochemistry studies in 30 clinical specimens revealed overexpression of CYP1A1 in various types of ovarian cancers compared with benign epithelia and frequent localization of the enzyme to cancer cell nuclei. Forced expression of CYP1A1wt or CYP1A1v in HOSE cells resulted in nuclear localization of the enzyme and acquisition of anchorage-independent growth, which was further exacerbated following exposure to benzo(a)pyrene or 17beta-estradiol. Collectively, these data provided the first evidence that CYP1A1 overexpression and alternative splicing could contribute to ovarian cancer initiation and progression.