<p> Breast cancer cell dormancy is a significant clinical problem which contributes to the development of distant metastasis and disease relapse. Currently, no therapies exist which can effectively detect or eradicate dormant cancer cells. </p><p> In this study, we utilized a 3D co-culture dormancy model, recapitulating the inhibitory hematopoietic stem cell niche, which interacts with MDA-MB-231 cells, causing them to enter a state of growth arrest. The knockdown of emerging dormancy regulator gene, p38/MAPK14, in MDA-MB-231 cells allows previously dormant cells to &ldquo;break&rdquo; dormancy and re-enter the cell cycle when grown in the inhibitory niche. Using the newly described in vitro dormancy model, we performed a genomic shRNA library screen, and identified several p38-regulated breast cancer dormancy suppressor gene candidates. Two p38-regulated gene candidates were investigated further. Knockdown of transcription factors and p38 substrates, HBP1 and BHLHB3, in MDA-MB-231 cells lead to re-activation (proliferation) of once indolent cells when cultured in the inhibitory niche. </p><p> The present study illustrates the role of p38 and p38-regulated genes in breast cancer dormancy within the microenvironment of the inhibitory (endosteal) hematopoietic stem cell niche. Additionally, we have identified a list of ~700 breast cancer dormancy suppressor candidate genes. Further analysis and validation experiments are needed to classify novel molecular players and signaling pathways involved in tumor cell dormancy from the list of candidate genes generated in this study.</p>

<p> Every year in the US, over 200,000 men are diagnosed with prostate cancer (PCa) and annual mortality in the US exceeds 30,000. PCa is treated surgically, and post-surgical recurrent tumors treated through targeted inhibition of the androgen receptor (AR) and AR signaling through disruption of androgen synthesis. If tumors resist antiandrogen therapy, castration recurrent (CR) tumors are treated with second line therapies that currently involve more potent inhibition of AR. Since the advent of these newer therapies, alternative, AR-independent resistance mechanisms have begun to become more prevalent. Thus, there is a need to explore and discover novel pathways, drug targets, and mechanisms driving aggressive and atypical subtypes of PCa. </p><p> Non-coding RNAs (ncRNAs) are known to play critical roles in normal cell behavior, as well as various diseases including cancer. Small nucleolar RNAs (snoRNAs) are one class of ncRNAs that are primarily involved in guiding specific enzymatic modifications (e.g. 2&rsquo;O-methylation, pseudouridylation) of ribosomal RNAs (rRNAs) and thereby allowing fidelity of ribosome biogenesis. HBII-52 is an orphan C/D box snoRNA encoded in tandem repeat copies at chromosomal locus 15q11-13. HBII-52 has no predicted or reported rRNA targets, however, it has both predicted and validated messenger RNA (mRNA) targets, including the serotonin 2c receptor (5-HT2cR, encoded by the gene HTR2C). 5-HT2cR is a G-Protein Coupled Receptor (GPCR) predominantly expressed in brain, and it controls appetite and signaling via cognate ligand, serotonin (5-HT) binding. The pre-mRNA encoding 5-HT2cR is subject to complex alternative processing including alternative pre-mRNA splicing and Adenosine-to-Inosine (A-to-I) RNA editing. HBII-52 promotes processing to, highly active isoforms of 5-HT2cR, thus potentiating its signaling axis. A variety of inhibitors of 5-HT2cR exist including potent and selective inhibitors like SB242,084 that have already undergone preclinical evaluation for neurologic disorders. </p><p> Herein, we report that the expression levels of HBII-52 (MBII-52 in mouse) and 5-HT2cR are deregulated in PCa including both mouse and human preclinical models, and human clinical specimens. Furthermore, mechanistic studies of the HBII-52/5-HT2cR pathway in PCa cells indicated this pathway drives transition to an aggressive and invasive phenotype, specifically by a highly active, less edited isoform of 5-HT2cR. Finally, we evaluated the feasibility and efficacy of targeting 5-HT2cR using small molecules in PCa and arrived at a lead drug candidate, SB242,084. In conclusion, active edit-isoforms of 5-HT2cR promote aggressiveness and invasiveness of PCa cells. One potential mode of activation includes upregulation of HBII-52. HBII-52/5-HT2cR-positive cancer cells can be effectively targeted through selective inhibition of 5-HT2cR with small molecules.</p>

No description available.

No description available.

Progression through the G1, S, G2 and M phases of the cell cycle is controlled by cyclin-dependent kinases (Cdks) and cyclins. These proteins form active Cdk:cyclin complexes that phosphorylate specific substrates. The Cdk:cyclin complexes of the G1/S transition regulate the progression of cells into the S phase by phosphorylating the retinoblastoma protein (Rb). This prevents Rb from sequestering E2F, a transcription factor that induces expression of genes required for DNA synthesis. This process is in part regulated by a family of Cdk inhibitors (CKIs) called the Ink4 family (Inhibitors of Cdk4). The Ink4 family of CKIs consists of four members; p16Ink4a, p15Ink4b, p18Ink4c and p19Ink4d, and they bind specifically to Cdk4 and Cdk6, thereby negatively regulating their kinase activities and cell cycle progression. Because of its cell cycle inhibitory role, p16Ink4a is frequently mutated or deleted in human cancer, whereas the other Ink4 genes are only occasionally altered in cancer. The overall aim of this thesis was to study the roles of p18Ink4c and p19Ink4d using in vivo models of cancer and embryonic development. In paper I, we analyzed the tumor spectrum in mice lacking p53, Ink4c and Ink4d. p53 is a tumor suppressor and one of the most frequently mutated genes in human cancer. Mice carrying mutated p53 alleles are highly tumor-prone but develop predominantly lymphomas. However, the combined loss of p53 and Ink4c (but not Ink4d) caused a shift in the tumor spectrum to increased incidences of hemangiomas and hemangiosarcomas, as well as appearance of medulloblastomas, a tumor of the cerebellum. These data, revealed in the absence of p53, suggest a cell-type specific tumor suppressing role for p18Ink4c. In paper II, loss of Ink4c was evaluated in another tumor-prone mouse model; the Eµ-Myc mouse. This is a transgenic mouse overexpressing c-Myc in B cells causing clonal B cell lymphomas. Surprisingly, precancerous B cells and lymphomas from Eµ-Myc mice exhibited elevated levels of p18Ink4c mRNA and protein despite high rates of proliferation. Moreover, loss of Ink4c in this model did not affect the rate of cell proliferation or the onset of tumor development. We conclude from these studies that Ink4c is not an important tumor suppressor of Myc-induced lymphomas. To gain insight into the role of Ink4 genes in early vertebrate development, the African clawed frog, Xenopus laevis, was analyzed for the presence of Ink4 homologs. Paper III describes the cloning and characterization of a gene homologous to Ink4d, Xl-Ink4d. This CKI is expressed throughout frog embryo development, making Xl-Ink4d the only CKI present during the cleavage stages of X. laevis. Antisense morpholino oligonucleotides directed against Xl-Ink4d were used to knock down the protein level of Xl-Ink4d during development. This resulted in defects in head tissues and reduced expression of Twist, a gene important for neural crest cell migration. We therefore propose that Xl-Ink4d is important for proper neural crest differentiation in the frog.

The Notch and TGF13 signaling pathways have been shown to play important roles in regulating endothelial-to-mesenchymal transition (EndMT) during cardiac morphogenesis. EndMT is the process by which endocardial cells of the atrioventricular canal and the outflow tract repress endothelial cell phenotype and upregulate mesenchymal cell phenotype. EndMT is initiated by inductive signals emanating from the overlying myocardium and inter-endothelial signals and generate the cells that form the heart valves and atrioventricular septum. The Notch and TGFf3 pathway are thought to act in parallel to modulate endothelial phenotype and promote EndMT. Vascular endothelial (VE) cadherin is a key regulator of cardiac endothelial cell phenotype and must be downregulated during EndMT. Accordingly, VE-cadherin expression remains stabilized in the atrioventricular canal and outflow tract of Notchl-deficient mouse embryos, while activation of the Notch or TGFP pathways results in decreased VE-cadherin expression in endothelial cells. However, the downstream target gene(s) that are involved in regulating endothelial cell phenotype and VE-cadherin expression remain largely unknown. In this thesis the transcriptional repressor Slug is demonstrated to be expressed by the mesenchymal cells and a subset of endocardial cells of the atrioventricular canal and outflowtract during cardiac morphogenesis. Slug is demonstrated to be required for cardiac development through its role in regulating EndMT in the cardiac cushion. Data presented in Chapter 6 further suggests that Slug-deficiency in the mouse is compensated for by a increase in Snail expression after embryonic day (E) 9.5, which restores EndMT in the cardiac cushions. Additionally, the Notch pathway, via CSL, directly binds and regulates expression of the Slug promoter, while a close Slug family member, Snail is regulated by the TGFB pathway in endothelial cells. While Notch does not directly regulate Snail expression, Notch and TGFB act synergistically to regulate Snail expression in endothelial cells. It is further demonstrated that Slug is required for Notch mediated EndMT, binds to and represses the VE-cadherin promoter, and induces a motile phenotype. Collectively the data demonstrate that Notch signaling directly regulates Slug, but not Snail, expression and that the combined expression of Slug and Snail are required for cardiac cushion morphogenesis.

<p> Copepod nauplii are important members of the marine planktonic community, and they can be the most abundant component of the microzooplankton. Despite the importance of copepod early life history stages to food web dynamics and carbon flux in the sea, there is a paucity of information about their ecology due to challenges in identifying nauplii to species, and in sampling them quantitatively. I report here on the development and optimization of a new molecular method that uses quantitative PCR (qPCR) to identify and estimate the abundances of nauplii of a common coastal copepod, <i>Parvocalanus crassirostris,</i> in field samples. The following experiments were performed towards this goal: I surveyed the genetic diversity of copepods in the study region, optimized sample treatment for qPCR, developed a size fractionation protocol to separate life stages of the target species, quantified the mitochondrial cytochrome C oxidase subunit I (mtCOI) gene copies in each <i> P. crassirostris</i> life stage, tested the effect of food levels on mtCOI copy number in nauplii, and compared direct counts to qPCR estimates of the target species to validate the qPCR method. The number of mtCOI gene copies in each developmental stage of this species was found to increase by &sim;1.5 orders of magnitude from early nauplii to adult. Food level experiments suggested that mtCOI copy number may be influenced by feeding environment in late naupliar stages. In validation experiments, qPCR estimates were 68 to 130% of the number estimated from direct counts. Both methods had a coefficient of variation of approximately 16%, indicating similar precision across methods. As a field test of the method, daily samples were collected in southern Kane&lsquo;ohe Bay and used to quantify the density <i>P. crassirostris</i> nauplii over a 13-day period in the summer of 2011. The average density of <i> P. crassirostris</i> nauplii in developmental stages NII - NV was found to be 1.5&times;10³ individuals m^-3 over the 13-day period. The qPCR-based method developed here will enable future studies on naupliar ecology in the field, including investigation of food web, population, and community dynamics.</p>

<p> A complex molecular network is put into place at specific phases of the cell cycle to prevent unscheduled cell division that could result in malignant cell growth. Emerging evidence shows that still uncharacterized proteins play crucial functions at those cell cycle transition points. Nuclear protein Msp58 and EDD E3 ubiquitin ligase have been implicated in different aspects of cell proliferation and reported to be abnormally expressed in numerous types of cancers. The molecular mechanisms underlying Msp58 and EDD functions, however, are not well understood. The work presented here shows that Msp58 and EDD form a stable protein complex that regulates cell viability and proliferation. Interestingly, knockdown of EDD by RNA interference leads to a significant accumulation of Msp58 protein, which suggests that EDD serves as a negative regulator of Msp58. In addition, our in vivo ubiquitination assays and analyses of various cell lines treated with translational and proteasomal inhibitors demonstrate that Msp58 is regulated post-translationally by the ubiquitin-proteasome pathway. These results imply that EDD ligase activity is involved in this regulatory process. Using flow cytometry analyses and biochemical characterization of Msp58 and/or EDD depleted cells, we show that the Msp58-EDD complex plays important roles in cell cycle progression via the control of cyclin gene expression. In particular, silencing Msp58 and/or EDD alters the protein levels of cyclins B, D and E. Taken together, our data suggest that a set of the biological roles attributed to Msp58 and EDD may be executed in the context of the complex that they form, thereby revealing a novel molecular mechanism for these two proteins to accomplish their functions.</p>

<p> Through the use of a novel immunodetection assay, our lab identified a series of mutants that internally accumulate the precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY), a phenotype that suggests a defect at the late e&barbelow;n&barbelow;dosome to v&barbelow;acuole (<i> ENV</i>) interface of the biosynthetic pathway. This study focuses on two of the novel genes identified: <i>ENV10</i> and <i>ENV11 </i> and is the first to establish the cellular localization of Env10p in the endoplasmic reticulum. Assays of vacuole and lipid droplet morphology, as well as growth characterization under various stressors demonstrate Env10p has a role in vacuolar protein trafficking and endomembrane system integrity and may operate in a parallel or compensatory manner to Env9p. This study also confirms that Env11p localizes to the nucleus in a saturable fashion where it may be involved in transcriptional regulation of genes involved in vacuole events in conjunction with Vid22p and Tbf1p.</p>

<p> <i>Ustilago maydis</i> is a basidiomycete fungus known to cause tumor formation in maize. It exhibits two distinct forms: yeast-like and filamentous. Both forms exhibit cell polarity, an inherent process observed in all cell types. In the Ascomycota, Tea1 homologs are involved in cell morphogenesis. Tea1 homologs had not been identified in the Basidiomycota. In this study, I have characterized the homolog of the <i>Schizosaccharomyces pombe tea1</i> gene in <i>U. maydis</i>. I have determined the &Delta;<i> tea1</i> null phenotype through a gene deletion strategy, established the subcellular localization of Tea1 and Tea4 using GFP fusions, and determined a Tea1 interaction with itself using a yeast two-hybrid approach. These studies provide support for a major role of Tea1 in cell morphogenesis, in maintaining an axis of polarized growth, and in cell polarity in <i>U. maydis</i>. </p>