Establishment of Rab-11 Induced Inflammatory Regulation as Therapeutic Targets in Colon Cancer Progression

Colon cancer is the third-deadliest cancer in the United States. Better understanding the cancer microenvironment/niches is crucial to the development of successful therapeutic targets. An RNAi screening using enterocyte specific driver was performed in Drosophila melanogaster intestine to search for niches regulating the intestine stem cell homeostasis. A small GTPase, Rab11 caused strong intestine stem cell (ISC) proliferation and tissue hyperplasia upon knockdown, due to increased production of inflammatory cytokines and growth factors. Increased inflammatory cytokines and proliferation were also observed in mouse Rab11a knockout (KO) intestine, indicating Rab11 regulatory role in the inflammation-induced hyperplasia is evolutionarily conserved and may also apply to human. We hypothesized that Rab11 is required to maintain cytokines in an appropriate state and its expression is down regulated in cancers. We investigated dextran sulfate sodium and chemical induced mouse colon cancer. Rab11 was largely reduced/absent in cancer tissues whereas well present in the normal tissue. We also investigated the correlation of Rab11 level and human cancer progression by immunofluorescence staining, and found that close to 50% and 40% of the cases studied had reduced Rab11 level by 20% and 30%, respectively. The greater the reduction is, the higher chance it is associated with more progressed cancer. Rab11, therefore, functions to suppress cancer progression and can be potentially developed towards a better diagnosis and treatment target for colon cancer. We will screen FDA approved drugs for ISC proliferation regulation, using a fly intestine tumor model established by expressing a human activated RAFGOFgene and a luciferase gene in the fly gut precursor cells. Selected drugs will be applied to test the Rab11 induced hyperplasia in fly, and further validated by mouse and human organoids derived from Rab11 KO mouse or human colon cancer tissues

TVIR: a comprehensive vegetable information resource database for comparative and functional genomic studies

Vegetables are an indispensable part of the daily diet of humans. Therefore, it is vital to systematically study the genomic data of vegetables and build a platform for data sharing and analysis. In this study, a comprehensive platform for vegetables with a user-friendly Web interface—The Vegetable Information Resource (TVIR, http://tvir.bio2db.com)—was built based on the genomes of 59 vegetables. TVIR database contains numerous important functional genes, including 5215 auxin genes, 2437 anthocyanin genes, 15 002 flowering genes, 79 830 resistance genes, and 2639 glucosinolate genes of 59 vegetables. In addition, 2597 N6-methyladenosine (m6A) genes were identified, including 513 writers, 1058 erasers, and 1026 readers. A total of 2 101 501 specific clustered regularly interspaced short palindromic repeat (CRISPR) guide sequences and 17 377 miRNAs were detected and deposited in TVIR database. Information on gene synteny, duplication, and orthologs is also provided for 59 vegetable species. TVIR database contains 2 346 850 gene annotations by the Swiss-Prot, TrEMBL, Gene Ontology (GO), Pfam, and Non-redundant (Nr) databases. Synteny, Primer Design, Blast, and JBrowse tools are provided to facilitate users in conducting comparative genomic analyses. This is the first large-scale collection of vegetable genomic data and bioinformatic analysis. All genome and gene sequences, annotations, and bioinformatic results can be easily downloaded from TVIR. Furthermore, transcriptome data of 98 vegetables have been collected and collated, and can be searched by species, tissues, or different growth stages. TVIR is expected to become a key hub for vegetable research globally. The database will be updated with newly assembled vegetable genomes and comparative genomic studies in the future

Functional comparison and analysis of protein-protein interactions of Autographa californica multiple nucleopolyhedrovirus transregulatory proteins IE0 and IE1

Autographa californica multiple nucleopolyhedrovirus expresses two major transregulatory proteins IE0 and IE1 immediately upon infection. IE0 differs from IE1 only by 54 additional N-terminal amino acids (aa). Either IE0 or IE1 can support viral replication; however both are required for a wild-type infection. It is unknown what the different functions of IE0 and IE1. Both IE0 and IE1 can transactivate viral early genes and support viral DNA replication. It is therefore hypothesized that by the addition of Nterminal 54 aa, IE0 acquires different transactivation activity on viral genes and interacts with different viral or host partners. To test this hypothesis, functional comparisons between IE0 and IE1 and the identification of their interaction partners in infected cells were performed. Comparisons of subcellular localization and transactivation activities between IE0 and IE1 showed no difference. However analyses of the nucleocapsid content of occlusion derived virions (ODV) revealed that IE0 and IE1 appear to regulate the number of nucleocapsids per ODV. Deletion within the IE0 specific N-terminal 54 aa did not affect IE0 transactivation dramatically but reduced its ability to support viral DNA replication. Analyses of interacting proteins did not identify any proteins that were specific to either with IE0 or IE1. However, the viral protein AC16 (BV/ODV-E26) was shown to bind to both IE0 and IE1 via a binding domain at IE1 72-99 aa. Mutation of the binding domain enhanced budded virus (BV) production by viruses expressing only IE0 but not IE1. Deletion of ac16 however resulted in increased levels of IE0 relative to IE1 as the only observable impact. These results would therefore indicate that AC16 regulates ie0 expression. Deletion of ac16 and the overlapping gene ac17, interestingly resulted in a significant delay of viral gene expression for up to 12 hours. However, the delay of viral gene expression was only observed with BV-infected cells and not in cells infected by transfecting viral DNA. AC16 and AC17 are therefore critical for rapid gene expression during the very early events of infection, and highlight the fact that proteins interacting with IE0 and IE1 play key roles in baculovirus biology.Land and Food Systems, Faculty ofGraduat

TLR sorting by Rab11 endosomes maintains intestinal epithelial-microbial homeostasis

Compartmentalization of Toll-like receptors (TLRs) in intestinal epithelial cells (IECs) regulates distinct immune responses to microbes; however, the specific cellular machinery that controls this mechanism has not been fully identified. Here we provide genetic evidences that the recycling endosomal compartment in enterocytes maintains a homeostatic TLR9 intracellular distribution, supporting mucosal tolerance to normal microbiota. Genetic ablation of a recycling endosome resident small GTPase, Rab11a, a gene adjacent to a Crohn\u27s disease risk locus, in mouse IECs and in Drosophila midgut caused epithelial cell-intrinsic cytokine production, inflammatory bowel phenotype, and early mortality. Unlike wild-type controls, germ-free Rab11a-deficient mouse intestines failed to tolerate the intraluminal stimulation of microbial agonists. Thus, Rab11a endosome controls intestinal host-microbial homeostasis at least partially via sorting TLRs

Recycling Endosomes in Mature Epithelia Restrain Tumorigenic Signaling

The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, whereas suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis. SIGNIFICANCE: Recycling endosome traffic in mature epithelia constitutes a novel tumor suppressing mechanism

Autographa californica Multiple Nucleopolyhedrovirus Core Gene ac96 Encodes a Per Os Infectivity Factor (pif-4)▿

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc96null). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc96null BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc96null was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4)

Gene expression profiling identifies the zinc-finger protein Charlatan as a regulator of intestinal stem cells in Drosophila

Intestinal stem cells (ISCs) in the adult Drosophila midgut can respond to tissue damage and support repair. We used genetic manipulation to increase the number of ISC-like cells in the adult midgut and performed gene expression profiling to identify potential ISC regulators. A detailed analysis of one of these potential regulators, the zinc-finger protein Charlatan, was carried out. MARCM clonal analysis and RNAi in precursor cells showed that loss of Chn function caused severe ISC division defects, including loss of EdU incorporation, phosphorylated histone 3 staining and expression of the mitotic protein Cdc2. Loss of Charlatan also led to a much reduced histone acetylation staining in precursor cells. Both the histone acetylation and ISC division defects could be rescued by the simultaneous decrease of the Histone Deacetylase 2. The overexpression of Charlatan blocked differentiation reversibly, but loss of Charlatan did not lead to automatic differentiation. The results together suggest that Charlatan does not simply act as an anti-differentiation factor but instead functions to maintain a chromatin structure that is compatible with stem cell properties, including proliferation