Category C. Cancer Biology / Molecular Biology
C1. Characterization of septate junction biogenesis during embryogenesis in Drosophila
Sonia Hall, Jennifer Mendez, Sam Long, and Robert Ward. Molecular Biosciences, University of Kansas, Lawrence, KS.
Polarized epithelial cells form tissues that provide a protective barrier from the outside environment and allow for the compartmentalization of internal organs. The ability of an epithelium to act as a barrier is a universal requirement for all multicellular organisms, and is mediated by the formation of cellular junctions along the lateral membranes between epithelial cells. Tight junctions serve this function in vertebrate organisms, whereas septate junctions (SJ) are used in invertebrate organisms. Although tight and septate junctions are ultrastructurally distinct, they share several molecular components including proteins of the claudin and MAGUK families. Genetic studies in Drosophila have identified more than 20 genes that function in the assembly or maintenance of SJ, and recent studies have begun to shed light on the process of biogenesis. Most SJ genes are zygotically expressed and initially trafficked to the basolateral membrane. Many of these proteins are subsequently endocytosed and retargeted to the apical lateral region. Mature SJ are composed of highly crosslinked protein complexes that show very little mobility in the plane of the membrane. Interestingly, SJ proteins exhibit an interdependence, in which the loss of one core protein results in the mislocalization and increased mobility of all other SJ proteins along the lateral membrane. We noticed that the degree of mislocalization of certain SJ proteins is dependent upon which core component is missing. We are therefore extending these studies by examining the localization of ~10 SJ proteins in more than 20 different mutant backgrounds. In addition, we are using immunohistochemistry to track the colocalization of many SJ proteins as they are trafficked to the SJ during its biogenesis in wild type cells. These analyses should allow us to make inferences about the substructure of SJs during their biogenesis and maintenance.
C2. Screening for inhibitors of RNA-binding protein Musashi-1
Lan Lan1, Carl Appelman1, Amber Smith1, Jia Yu1, Rebecca Marquez1, Philip Gao2, Na Zhang2, Anuradha Roy3, Asokan Anbanandam4, Ragul Gowthaman1,5, John Karanicolas1,5, Jeffrey Aubé6, Kristi Neufeld1, and Liang Xu1
1Department of Molecular Biosciences, 2Protein Production Group, NIH COBRE in Protein Structure and Function, 3High Throughput Screening Laboratory, 4Bio-NMR Core Facility, 5Center for Bioinformatics, 6Department of Medicinal Chemistry, The University of Kansas.
The Notch signaling pathway is involved in several important cellular processes, such as cell proliferation, differentiation, and apoptosis. Dysregulation of Notch signaling can enhance tumorigenesis, by stimulating proliferation and maintenance of a small population of cancer cells, called cancer stem cells. Another major signaling pathway, Wnt signaling pathway, has been shown to have cross-talk between Notch pathway in cancer development. An important regulator of Notch signaling is Musashi-1 (Msi1). Msi1 negatively regulates expression of Numb, a protein that inhibits Notch signaling. Msi1 binds to the 3’ untranslated region of Numb mRNA, preventing translation of Numb protein, which results in de-repression of Notch signaling. Our hypothesis is that small molecules that disrupt the Msi1-Numb binding would result in down regulation of Notch signaling, and therefore inhibit cancer stem cell maintenance and self-renewal. Using in vitro fluorescence polarization (FP) assay, we have screened chemical libraries and identified a series of Msi1 inhibitors with sub-micromolar inhibition constants (Ki). The hits are confirmed through orthogonal SPR and NMR assays. These Msi1 inhibitors may potentially inhibit cancer cell growth by inhibiting Wnt- and Notch-pathways. By combining the NMR data of our lead compounds with computational efforts, we have developed structural models of Msi1 bound to these inhibitors and identified a new series of Msi1 inhibitors. These studies will provide a stepping stone for future validation assays in vitro and in vivo. The long term goal is to discover Msi1 inhibitors with hopes of inhibiting Notch/Wnt signaling pathways and thereby inhibiting cancer stem cell maintenance and self-renewal.
C3. Mutagenesis screen to identify genes involved in tissues specific growth of the larval trachea in Drosophila melanogaster
Paulo Leal and Robert Ward
Department of Molecular Biosciences, University of Kansas
During post-embryonic development in animals, different tissues and organs grow at different rates relative to each other, likely tied to each organ’s unique requirements during development and homeostasis. Differential growth occurs in spite of the fact that overall growth is tied to nutrition, which is largely regulated through the insulin signaling pathway. This suggests that tissue-specific mechanisms function in concert with or in parallel to insulin signaling to control their post-embryonic growth. One way to understand these mechanisms is through the characterization of mutations that specifically alter growth in single organs or tissues in a genetically tractable model system. The larval trachea of Drosophila is suited for this study as it is a well-studied branched tubular organ required for gas exchange that grows dramatically during larval development. Upon hatching, however, the larva begins to feed and thus organ growth is tied to nutrition. Mutations in two genes, uninflatable (uif) and Matrix metalloproteinase 1 (Mmp1) have phenotypes that include tissue specific growth reductions within the larval trachea. To identify additional genes that regulate larval tracheal growth, we are screening two collections of late larval/pupal lethal mutations: 49 P-element lines obtained from the Bloomington stock center and 252 EMS induced lines from the collection of 3rd chromosome late lethals generated by Dr. Bashirullah (University of Wisconsin). Preliminary screening identified 2 P-element and 4 EMS mutations that show specific larval tracheal defects. We are mapping and conducting phenotypic analysis on the mutant larvae, and examining interactions between uif, Mmp1, and the isolated mutants.
C4. MicroRNA regulation of Musashi-1 in colon cancer
Amber Smith, Rebecca Marquez, Lan Lan, Bryan Tsao, Xiaojie Meng, Kristi Neufeld, Liang Xu
Department of Molecular Biosciences, University of Kansas, Lawrence, KS
Musashi-1 (Msi1) is an RNA binding protein that inhibits translation of target mRNAs: APC, mNumb and p21WAF-1. By down-regulating mNumb and APC, Msi1 positively regulates the Notch and Wnt signaling pathways. A recent study discovered an over-expression of Msi1 in 67% of colorectal cancer patient samples as compared to paired healthy tissue. Furthermore, Msi1 is up-regulated in breast, medulloblastoma and glioblastoma cancers. The mechanisms responsible for negatively regulating Msi1 have yet to be discovered. MicroRNAs (miRNAs), 20-22 nucleotide RNAs, regulate genes post-transcriptionally by binding to the 3’ untranslated region (UTR) of target mRNAs and repressing translation. Recently, studies demonstrate that certain miRNAs regulate Msi1 expression in glioblastoma cells. The purpose of this study is to learn more about the effects of miRNA-mediated down-regulation of Msi1 in order to exploit this interaction as a novel miRNA-based molecular therapy in colon and breast cancer. Utilizing miRNA prediction programs, we identified miR-137 as possessing binding sites within the Msi1 3’-UTR. Using quantitative PCR (qPCR), we found an inverse correlation between miR-137 and Msi1 expression in colon cancer cell lines. Restoration of miR-137 in colon cancer cells decreased Msi1 expression and increased translation of Msi1 targets, mNumb and p21WAF-1. Furthermore, upon miR-137 restoration, Notch and Wnt signaling was reduced along with inhibition of cell proliferation, colony formation and tumorsphere growth in cancer cell lines with high Msi1. The outcome of this study portrays miR-137 as a tumor suppressive miRNA through the negative regulation of oncogenic Msi1.