FUNCTION OF ZNF668 IN CANCER DEVELOPMENT

Human cancer develops as a result of accumulation of mutations in oncogenes and tumor suppressor genes. Zinc finger protein 668 (ZNF668) has recently been identified and validated as one of the highly mutated genes in breast cancer, but its function is entirely unknown. Here, we report two major functions of ZNF668 in cancer development. (1) ZNF668 functions as a tumor suppressor by regulating p53 protein stability and function. We demonstrate that ZNF668 is a nucleolar protein that physically interacts with both MDM2 and p53. By binding to MDM2, ZNF668 regulates MDM2 autoubiquitination and prevents MDM2-mediated p53 ubiquitination and degradation; ZNF668 deficiency impairs DNA damage-induced p53 stabilization. Notably, ZNF668 effectively suppresses breast cancer cell proliferation and transformation in vitro and tumorigenicity in vivo. Consistently, ZNF668 knockdown readily transforms normal mammary epithelial cells. Together, our studies identify ZNF668 as a novel breast tumor suppressor gene that acts at least in part by regulating the stability and function of p53. (2) ZNF668 functions as a DNA repair protein by regulating histone acetylation. DNA repair proteins need to access the chromatin by chromatin modification or remodeling to use DNA template within chromatin. Dynamic posttranslational modifications of histones are critical for cells to relax chromatin in DNA repair. However, the precise underlying mechanism mediating enzymes responsible for these modifications and their recruitment to DNA lesions remains poorly understood. We observed ZNF668 depletion causes impaired chromatin relaxation as a result of impaired DNA-damage induced histone H2AX hyper-acetylation. This results in the decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after DNA damage, albeit with the presence of a functional ATM/ATR dependent DNA-damage signaling cascade. Importantly, the impaired loading of repair proteins and the defect in DNA repair in ZNF668-deficient cells can be counteracted by chromatin relaxation, indicating that the DNA-repair defect that was observed in the absence of ZNF668 is due to impeded chromatin accessibility at sites of DNA breaks. Our findings therefore identify ZNF668 as a key molecule that links chromatin relaxation with response to DNA damage in the control of DNA repair.

Regulation and function of {\it Xparaxis\/} during the development of paraxial mesoderm in {\it Xenopus laevis\/}

Genes of the basic helix-loop-helix transcription factor family have been implicated in many different developmental processes from neurogenesis to myogenesis. The recently cloned bHLH transcription factor, paraxis, has been found to be expressed in the paraxial mesoderm of the mouse suggesting a role for paraxis in the development of this mesodermal subtype which gives rise to the axial muscle, skeleton, and dermis of the embryo. In order to perform in vivo gain of function assays and obtain a better understanding of the possible roles of paraxis in mesodermal and somitic development, we have successfully identified homologues of paraxis in the frog, Xenopus laevis, where the process of mesodermal induction and development is best understood. The two homologues, Xparaxis-a and Xparaxis-b, are conserved with respect to their murine homologue in structure and expression within the embryo. Xparaxis genes are expressed immediately after gastrulation in the paraxial mesoderm of Xenopus embryos and are down regulated in the myotome of the mature somite with continued expression in the undifferentiated dermatome. Overexpression of Xparaxis-b in Xenopus embryos caused defects in the organization and morphology of the somites. This effect was not dependent on DNA binding of Xparaxis but is likely due to its dimerization with other bHLH factors. Co-injections with XE12 did not diminish the effects indicating that the defects were not the result of limiting amounts of XE12. We also demonstrated that Xparaxis does not cause obvious defects in the cell adhesions and movements required for proper mesoderm patterning during gastrulation. The paraxis proteins also lacked the ability to activate transcription as GAL4 fusion proteins in a GAL4 reporter assay, indicating that the genes may function more as modulators of the activity of dimerization partners than as positively acting cell determination factors. In agreement with this, Xparaxis is regulated in response to other pathways of bHLH gene action, in that XE12 can activate Xparaxis-b, in vivo. In addition we show regulation of Xparaxis in response to mMyoD induced myogenesis pathways, again suggesting Xparaxis plays an important role in the patterning and organization of the paraxial mesoderm. ^

The function of math5 and myocilin in mammalian eye development and disease

Complex molecular events underlie vertebrate eye development and disease. The eye is composed of two major tissue types: the anterior and posterior segments. During development, the retinal progenitor cells differentiate into six neuronal and one non-neuronal cell types. These cell types later organize into the distinct laminar structure of the mature retina which occupies the posterior segment. In the developed anterior segment, both the ciliary body and trabecular meshwork regulate intraocular pressure created by the aqueous humor. The disruption in intraocular pressure can lead to a blinding condition called glaucoma. To characterize molecular mechanisms governing retinal development and glaucoma, two separate mouse knockout lines carrying mutations in math5 and myocilin were subjected to a series of in vivo analyses. ^ Math5 is a murine homologue of Drosophila atonal , a bHLH proneural gene essential for the formation of photoreceptor cells. The expression of math5 coincides with the onset of retinal ganglion cell differentiation. The targeted deletion of mouse math5 revealed that a null mutation inhibits the formation of a majority of the retinal ganglion cells. The mutation also interferes with the normal development of other retinal cell types such as amacrine, bipolar and photoreceptor cells. These results suggest that math5 is a proneural gene responsible for differentiation of retinal ganglion cells and may also have a role in normal development of other neuronal cell types within the retina. ^ Myocilin has two unique protein coding regions bearing homology to non-muscle myosin of Dictyostelium discoideum and to olfactomedin, an extracellular matrix molecule first described in the olfactory epithelium of the bullfrog. Recently, autosomal dominant forms of myocilin mutations have been found in individuals with primary open-angle glaucoma. The genetic linkage to glaucoma suggests a role of myocilin in normal intraocular pressure and ocular function. However, the analysis of mice heterozygous and homozygous for a targeted null mutation in myocilin indicates that it is dispensable for normal intraocular pressure or ocular function. Additionally, the lack of a discernable phenotype in both heterozygous and null mice suggests that haploinsufficiency is not a critical mechanism for MYOC-associated glaucoma in humans. Instead, disease-causing mutations likely act by gain of function. ^ In summary, these studies provide novel insights into the embryonic development of the vertebrate retina, and also begin to uncover the molecular mechanisms responsible for the pathogenesis of glaucoma. ^

Analyzing the function of myogenin in adult satellite cells through the construction of a myogenin conditional allele

Although mechanisms regulating the formation of embryonic skeletal muscle are well characterized, less is known about muscle formation in postnatal life. This disparity is unfortunate because the largest increases in skeletal muscle mass occur after birth. Adult muscle stem cells (satellite cells) appear to recapitulate the events that occur in embryonic myoblasts. In particular, the myogenic basic helix-loop-helix factors, which have crucial functions in embryonic muscle development, are assumed to have similar roles in postnatal muscle formation. Here, I test this assumption by determining the role of the myogenic regulator myogenin in postnatal life. Myogenin-null mice die at birth, necessitating the generation of floxed alleles of myogenin and the use of cre-recombinase lines to delete myogenin. Removing myogenin before embryonic muscle development resulted in myofiber deficiencies identical to those observed in myogenin-null mice. However, mice in which myogenin was deleted following embryonic muscle development had normal skeletal muscle, except for modest alterations in MRF4 and MyoD expression. Notably, myogenin-deleted mice were 30% smaller than controls, suggesting that myogenin's absence disrupted general body growth. These results suggest that skeletal muscle growth in postnatal life is controlled by mechanisms distinct from those occurring in embryonic muscle development. ^

The emergence of the social determinants of health on the policy agenda in Britain: A case study, 1980--2003

Numerous theories have been advanced in the effort to explain how a given policy issue manages to take root in the public sphere and subsequently move forward on the public legislative agenda—or not. This study examined how the social determinants of health (SDOH) came to be part of the legislative policy agenda in Britain from 1980 to 2003. ^ The specific objectives of the research were: (1) to conduct a sociopolitical analysis grounded in alternative agenda-setting theories to identify the factors responsible for moving the social determinants health perspective onto the British policy agenda; and (2) to determine which of the theories and related dimensions best accounted for the emergence of this perspective. ^ A triangulated content and context analysis of British news articles, historical accounts, and research commentaries of the SDOH movement was conducted guided by relevant agenda-setting theories set within a social movement framework to chronicle the emergence of the SDOH as a significant policy issue in Britain. ^ The most influential social movement and agenda setting elements in the emergence of the SDOH in Britain were issue generation tactics, framing efforts, mobilizing structures, and political opportunities grounded in social movement and agenda setting theories. Policy content or the details of the policy had comparatively little impact on the successful emergence of the SDOH. Despite resistance by the government, from 1980 to 1996 interest groups created a political understanding of the SDOH utilizing a framing package encompassing notions of inequality, fairness, and justice. This frame transmitted a powerful idea connected to a core set of British values and beliefs. After 1996, a shift in political opportunities cemented the institutional arrangements needed to sustain an environment conducive to the development and implementation of SDOH policies and programs. ^ This research demonstrates that the U.S. emergence of the SDOH on the policy agenda will depend upon: (1) U.S. ideals and values regarding poverty, inequality, race, health, and health care that will determine issue framing; (2) political opportunities that will emerge—or not—to advance the SDOH policy agenda; and (3) the mobilizing structures that support or oppose the issue. ^

The social construction of infant mortality: From grassroots to medicalization

Infant mortality as a problematic situation has been recognized for some 130 years in one form or another. It has undergone various changes in its empirical dimensions relative to whom we study within the population, what we study--low birth-weight vs. pre-term births--and how we study it--whether demographically or medically. An analysis of the process by which the condition was raised by claims makers as an intolerable situation among America's urban residents reveals that demographic and medical data were sparse. Nonetheless, a judgement about the meaning and significance of the condition was made, and that interpretation led to the promulgation of systems to both document and address the condition as it has come to be defined.^ This investigation depicts the historical context and natural history of infant mortality as one of a number of social problems that came to be defined through the interplay among groups and individuals making claims and how their claims came to the public policy agenda as worthy of collective resources--who won, who lost and why. The process of social definition focuses attention on the claims makers and the ways they contrast the meaning, origins and remedies for this troubling condition. The historical context becomes the frame of reference for understanding the actions of the claims makers and the meaning and significance they attached to the problem.^ We purport that "context" provides a closer reality than disjoined "value free" accounts. Context provides the evidence for the definition, who participated in the process, why and by what means.^ The role of women in the definitional process reveals the differences in approaches utilized by the women of the settlement house reform movement and African-American women working at the grass-roots. Much of the work done by these two groups provided options to the problem's remedy; however, their differences paved the way to our current (principally medically-oriented) definition and its inherent limitations. ^

A critical assessment of the National Institutes of Health Consensus Development Program against the eras which shaped it

This research study offers a critical assessment of NIH's Consensus Development Program (CDP), focusing upon its historical and valuative bases and its institutionalization in response to social and political forces. The analysis encompasses systems-level, as well as interpersonal factors in the adoption of consensus as the mechanism for resolving scientific controversies in clinical practice application. Further, the evolution of the CDP is also considered from an ecological perspective as a reasoned adaptation by NIH to pressures from its supporters and clients for translating biomedical research into medical practice. The assessment examines federal science policy and institutional designs for the inclusion of the public interest and democratic deliberation.^ The study relies on three distinct approaches to social research. Conventional historical methods were utilized in the interpretation of social and political influences across eras on the evolution of the National Institutes of Health and its response to demands for accountability and relevance through its Consensus Development Program. An embedded single-case study was utilized for an empirical examination of the CDP mechanism through five exemplar conferences. Lastly, a sociohistorical approach was taken to the CDP in order to consider its responsiveness to the values of the eras which created and shaped it. An exploration of organizational behavior with considerations for institutional reform as a response to continuing political and social pressure, it is a study of organizational birth, growth, and response to demands from its environment. The study has explanatory import in its attempt to account for the creation, timing, and form of the CDP, relative to political, institutional, and cultural pressures, and predictive import thorough its historical view which provides a basis for informed speculation on the playing out of tensions between extramural and intermural scientists and the current demands for health care reform. ^

A STUDY ON THE FUNCTION OF 14-3-3SIGMA IN REGULATING CANCER ENERGY METABOLISM

Metabolic reprogramming has been shown to be a major cancer hallmark providing tumor cells with significant advantages for survival, proliferation, growth, metastasis and resistance against anti-cancer therapies. Glycolysis, glutaminolysis and mitochondrial biogenesis are among the most essential cancer metabolic alterations because these pathways provide cancer cells with not only energy but also crucial metabolites to support large-scale biosynthesis, rapid proliferation and tumorigenesis. In this study, we find that 14-3-3σ suppresses all these three metabolic processes by promoting the degradation of their main driver, c-Myc. In fact, 14-3-3s significantly enhances c-Myc poly-ubiquitination and subsequent degradation, reduces c-Myc transcriptional activity, and down-regulates c-Myc-induced metabolic target genes expression. Therefore, 14-3-3σ remarkably blocks glycolysis, decreases glutaminolysis and diminishes mitochondrial mass of cancer cells both in vitro and in vivo, thereby severely suppressing cancer bioenergetics and metabolism. As a result, a high level of 14-3-3σ in tumors is strongly associated with increased breast cancer patients’ overall and metastasis-free survival as well as better clinical outcomes. Thus, this study reveals a new role for 14-3-3s as a significant regulator of cancer bioenergetics and a promising target for the development of anti-cancer metabolism therapies.

Defective antitumor function of monocyte -derived macrophages from epithelial ovarian cancer patients

An abundance of monocytes and macrophages (MO/MA) in the microenvironment of epithelial ovarian cancer (EOC) suggests possible dual roles for these cells. Certain MO/MA subpopulations may inhibit tumor growth by antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or stimulation of adaptive immunity. In contrast, other MO/MA subpopulations may support tumor growth by immunosuppressive or pro-angiogenic cytokine production. A better understanding of the phenotype and activity of MO/MA in EOC should lead to greater insight into their role in the immunopathobiology of EOC and hence suggest targets for treatment. We have found differences in the proportions of MO/MA subpopulations in the peripheral blood and ascites of EOC patients compared to normal donors, and differences in MO/MA surface phenotype in the associated tumor environment compared to the systemic circulation. We also demonstrate that, following their activation in vitro, monocyte-derived macrophages (MDM) from the peripheral blood and ascites of EOC patients exhibit antitumor effector activities that are different from the behavior of normal donor cells. The phenotypic characteristics and antitumor activity of CD14+ MO/MA and an isolated subpopulation of CD14brightCD16 −HLA-DR+ MO/MA were compared in samples of normal donor peripheral blood and the peripheral blood and ascites from EOC patients. MDM were cultured with macrophage colony-stimulating factor (M-CSF) and activated with lipopolysaccharide (LPS) or a combination of LPS plus recombinant interferon-gamma. We determined that MO/MA from EOC patients had altered morphology and significantly less ADCC and phagocytic activity than did MO/MA from normal donors. ADCC and phagocytosis are mediated by receptors for the Fe portion of IgG (FcγRs), the expression of which were also found to be deficient on EOC MDM from peripheral blood and ascites. Anti-tumor functions not mediated by the FcγRs, such as macrophage mediated cytotoxicity and cytostasis, were not impaired in EOC MDM compared to normal donor MDM. Our findings also showed that MDM from both EOC patients and normal donors produce M-CSF-stimulated cytokines, including interleukin-8, tumor necrosis factor alpha, and interleukin-6, which have the potential to support ovarian tumor growth and metastasis. These findings may be relevant to the pathogenesis of EOC and to the development of future bioimmunotherapeutic strategies. ^