Adrenocorticotropin Hormone Expression in the Developing Chicken Limb

In previous studies using mammalian models we have found both clinical and laboratory evidence of a role for melanocortins in endochondral ossification. The melanocortin system has remarkable conservation among vertebrates and melanocortin receptors are expressed with significant sequence homology in teleosts to mammals. The overall goal of these studies is to provide a more accessible model of melanocortin involvement in endochondral growth. We hope to determine if melanocortins play a role during endochondral ossification of the developing chicken limb. Like in mammals melanocortins are widely distributed throughout the body of chicken and participate in a wide range of physiological functions with the peripheral tissue distribution of melanocortin receptors in chicken more widespread. Our first step was to examine melanocortin expression in the developing limbs of the chick embryo. Using immunohistochemistry techniques, we detected ACTH (1-24) in the limbs of embryonic day 9 chick embryos. This initial data indicates that the chick embryo is a viable model that can be used to determine a role for melanocortin in endochondral growth. Melanocortin expression shows remarkable sequence homology, therefore results of these studies can be extrapolated to many vertebrate models

Stress Axis Hormones Induce Triglyceride Filled Nodule Formation in Vascular Smooth Muscle Cells

Homeostatic stress, such as that which occurs in diabetes, is associated with increased risk for the development of atherosclerosis. Atherosclerotic plaques of the artery wall are associated with both lipid accumulation and fibrous and/or calcified tissue accumulation. Vascular smooth muscle cells (VSMC) are derived from mesenchymal stem cells (MSC) which are capable of differentiating into adipocytes, chondrocytes and osteoblasts. MSC of the bone marrow are pushed toward the chondrogenic and adipogenic phenotypes in the presence of the stress hormones glucocorticoid and adrenocorticotropin (ACTH). This led us to hypothesize that the proliferative VSMC of the Goto-Kakizaki (GK) diabetic rat, when exposed to stress hormones will present an adipocytic and/or chondrogenic-like phenotype. VSMC of the GK rat were cultured using conditions that favor the multi-potential differentiation of MSC and were either left untreated, were treated with ACTH, dexamethasone (DEXA) or both. Cells were stained for lipid using oil-red-o, proteoglycan matrix using alcian blue and cell density using methylene blue. DEXA increased lipid nodule formation above the untreated control but the combined ACTH and DEXA treatment led to a significant increase above DEXA alone (lipid nodule #’s per field, DEXA 2.56 ± 1.63 vs. A+D 6.67 ± 1.68). These data suggest that stress hormones may contribute to VSMC matrix accumulation and lipid production during atherosclerosis development in diabetes

Abstract 3144: A role for DOK2 methylation in platinum resistance and tumor suppression in ovarian cancer

Ovarian cancer is the 5th leading cause of cancer in women, affecting close to 22,000 women in the year 2011, of which nearly 15,500 will die. It is difficult to detect until it reaches advanced stages and becomes malignant. Currently, the standard treatment for ovarian cancer is platinum-based therapeutics, such as Carboplatin or Cisplatin, combined with Taxol. Unfortunately, approximately 25% of patients are inherently platinum-resistant and all patients who suffer from recurrence will have developed acquired platinum resistance. The genetic/epigenetic causes of this resistance are poorly understood. Epigenetic events are reversible and the identification of genes altered by this mechanism may lead to studies on how to reprogram the process leading up to resistance. To examine the ovarian epigenome, we utilized an array based method, Methylation Oligonucleotide Microarray Analysis (MOMA), to analyze a set of 50 primary ovarian tumors and 12 ovarian normal samples. We identified epigenetic differences that segregated with platinum response and then associated this with expression data to identify gene candidates transcriptionally repressed and methylated in patients resistant to platinum. Next, a pooled shRNA screen was performed on candidate genes to identify those that were functionally relevant to platinum resistance. One of the validated candidate genes identified through the pooled shRNA screen was DOK2, an adapter protein downstream of tyrosine kinase, which has been shown by others to be a lung cancer tumor suppressor. We show that suppression of DOK2 by short hairpin RNAs in ovarian cell lines conferred resistance to platinum treatment. To elucidate the mechanism for resistance, we measured the influx of platinum into the cells using C-14 tagged carboplatin. As a result, uptake of carboplatin was found to be decreased with DOK2 suppression. Consistent with DOK2 having tumor suppressor activity, knockdowns in ovarian cell lines increases growth and migration. Furthermore, loss of DOK2 induces invasive and tumorigenic phenotypes in ovarian cell lines. DOK2 is already a proven tumor suppressor in lung cancer, and our experiments indicate DOK2 has tumor suppressor features in ovarian cancer as well. We show that DOK2 is a tumor suppressor in ovarian cancer and that the loss of DOK2 also contributes to platinum resistance. Understanding DOK2 function will help us understand ovarian cancer development, progression as well as therapy resistance

Abstract 70: A role for the chromatin remodeling protein CHD3 in ovarian cancer therapy response

Carboplatin and cisplatin are chemotherapeutic agents that are used extensively for treating epithelial ovarian cancer. These drugs can be highly effective, yet tumors are frequently refractory to treatment or become resistant upon tumor relapse. Epigenetic silencing, particularly at promoter regions of genes regulates important cell function and has been associated with all stages of tumor formation and progression and may contribute to therapy response. We analyzed the epigenome of 50 primary ovarian tumors and 12 normal ovarian samples using an array based method previously developed in our lab and associated Affymetrix U133 expression data. We then identified gene candidates that segregate patients based on platinum sensitivity and patient survival. These candidates were then pooled into a genome-wide RNAi-based screen where we validated a gene encoding a chromatin remodeling protein, CHD3, a member of the Mi-2 NuRD complex, and show that it is linked to chemoresistance. CHD3 is silenced through an epigenetic mechanism in both ovarian cancer cell lines and primary ovarian tumors. When ovarian cancer cell lines that are transcriptionally silenced for CHD3 are challenged with carboplatin they display a striking slow growth phenotype as well as increased resistance to the chemotherapy drugs carboplatin and cisplatin. Taken together, we provide the first evidence for a role for CHD3 as an important mediator of chemoresistance in ovarian cancer. Furthermore, CHD3 might represent a response predictor and potential therapeutic target for predicting chemoresistance in this disease

Abstract 2988: DOK2 suppression by methylation induces platinum resistance via suppression of apoptosis in ovarian cancer cells.

Ovarian cancers are highly heterogeneous and while chemotherapy is the preferred treatment, many patients are intrinsically resistant or quickly develop resistance. Furthermore, all tumors that recur will become resistant. Recent evidence suggests that epigenetic deregulation may be a key factor in the onset and maintenance of chemoresistance. To examine the ovarian epigenome, we first analyzed a set of 43 primary ovarian tumors and 9 normal ovarian samples. Since therapy response is a significant issue for ovarian cancer patients we analyzed the epigenetic differences that segregate with platinum response. We then associated expression data to identify genes with expression changes potentially altered by promoter methylation to generate a list of candidate platinum resistance genes. Next, we developed a tissue culture carboplatin resistance screen to determine which candidates functionally affect resistance. The screen utilized pools of shRNAs of the candidate genes to identify genes that when repressed allowed survival from carboplatin treatment, in order to validate that our epigenetics screen identified genes involved in resistance. Of the genes identified in the screen we further characterized one gene, docking protein 2 (DOK2), an adapter protein downstream of tyrosine kinase, to determine if we could elucidate what mechanism was used to increase resistance. Our analysis determined that loss of DOK2 decreased the level of apoptosis in response to carboplatin. In addition, we determined that in cells with reduced DOK2, the level of anoikis was decreased, a mechanism of possible importance in ovarian cancer where there is a large number of cells floating in ascites. Functional analysis of the DOK2 genes ability to affect resistance validates this approach to finding genes involved in carboplatin resistance

Abstract 1047: Suppression of the chromatin remodeling protein CHD3 and platinum resistance in ovarian cancer

Epithelial ovarian cancer is the leading cause of death from gynecological malignancies. Currently platinum-based chemotherapy, coupled with a taxane based drug is the primary treatment for ovarian cancer. Approximately 25% of patients either present with or rapidly develop resistance to platinum based chemotherapy and all recurrent tumors ultimately become resistant. Epigenetic modifications have been associated with tumor formation and progression and may contribute to therapy response. We hypothesize that Epigenetic changes such as DNA CpG methylation is in part responsible for the onset of chemoresistance of EOC. To identify epigenetically regulated genes associated with ovarian cancer chemotherapy resistance, a genome wide approach was used. For the most significant genes an in vitro culture system was developed to study platinum resistance. Candidate genes were screened by addition of shRNAs to model the transcriptional suppression caused by DNA methylation and genes that scored positive for increasing resistance were identified, one of them being the CHD3 gene. Here we show that loss of expression of CHD3, a member of the Mi-2/NuRD complex, causes increased resistance to platinum chemotherapy drugs. Additionally, ovarian cell lines transcriptionally silenced for CHD3 are more invasive, and have increased migratory ability. Recent evidence suggests molecular and phenotypic associations between chemo resistance and the acquisition of epithelial-mesenchymal transition of cancer cells. The transition of epithelial cell to a mesenchymal cell requires an alteration in morphology, cellular architecture, adhesion, and migration capacity. Cancer cells undergoing EMT can acquire invasive properties and enter the surrounding stroma, resulting in the creation of a favorable microenvironment for cancer progression and metastasis. Our results indicate that when CHD3 is silenced, cells undergo an EMT-like transition thereby allowing them to bypass apoptosis and resist platinum based therapy. Taken together, we provide the first evidence of a role for CHD3 as an important epigenetic regulator of chemoresistance in ovarian cancer and hypothesize EMT as one of the underlying mechanisms. Furthermore, CHD3 expression might represent a therapy response predictor and could be a future therapeutic target for ovarian cancer

Continent-wide analysis of how urbanization affects bird-window collision mortality in North America

Characteristics of buildings and land cover surrounding buildings influence the number of bird-window collisions, yet little is known about whether bird-window collisions are associated with urbanization at large spatial scales. We initiated a continent-wide study in North America to assess how bird-window collision mortality is influenced by building characteristics, landscaping around buildings, and regional urbanization. In autumn 2014, researchers at 40 sites (N = 281 buildings) used standardized protocols to document collision mortality of birds, evaluate building characteristics, and measure local land cover and regional urbanization. Overall, 324 bird carcasses were observed (range = 0–34 per site) representing 71 species. Consistent with previous studies, we found that building size had a strong positive effect on bird-window collision mortality, but the strength of the effect on mortality depended on regional urbanization. The positive relationship between collision mortality and building size was greatest at large buildings in regions of low urbanization, locally extensive lawns, and low-density structures. Collision mortality was consistently low for small buildings, regardless of large-scale urbanization. The mechanisms shaping broad-scale variation in collision mortality during seasonal migration may be related to habitat selection at a hierarchy of scales and behavioral divergence between urban and rural bird populations. These results suggest that collision prevention measures should be prioritized at large buildings in regions of low urbanization throughout North America