Category C. Cancer Biology/ Molecular Biology /Developmental Biology
C1. Septate Junction Genes are Essential during Drosophila Melanogaster Oogenesis
Haifa A. Alhadyian and Robert E. Ward, IV
Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
Morphogenesis is a highly regulated process that contributes to animal’s development in events such as gastrulation and neurulation. Morphogenesis is mainly accomplished by coordinated changes in cell shape and arrangement that are regulated by cell signaling events. During this process, cells within tissues are physically connected to each other through intercellular junctions that reside along the lateral plasma membrane of the cells. The apical adhesive molecule E-cadherin is critical for maintaining cell-cell attachment during morphogenesis, as mutation in E-cadherin causes severe developmental defects and lead to embryonic lethality. However, it is unclear if these interactions are sufficient for sustaining tissue integrity or whether other adhesive complexes are required as well. In invertebrate epithelia, septate junctions (SJs), functionally homologous to tight junctions in vertebrates, reside just basal to the adherens junction, and contain many adhesion molecules. Our preliminary data indicates that SJ genes are required for a number of morphogenetic events during embryonic and adult development in the fruit fly, Drosophila melanogaster. To determine the molecular mechanisms underlying the importance of SJ proteins during morphogenesis, we are investigating their roles during Drosophila oogenesis, specifically in a morphogenetic process called follicle cell rotation. The newly formed Drosophila egg chamber has a spherical-like structure that elongates coincident with global tissue rotation which begins shortly after egg formation. We found that the transmembrane protein Macroglobuin complement-related (Mcr), the cytoplasmic protein Coracle (Cor) and the cell adhesion protein Neuroglian (Nrg) are essential for egg chamber morphogenesis, as SJ mutant egg chambers failed to elongate. In addition, knocking down these SJ proteins results in missing middle stages egg chambers. Together, these observations suggest that SJ proteins are essential for egg chamber morphology during oogenesis as well as throughout the fruit fly development. Currently, we are examining the molecular mechanisms underlying the requirements of SJ proteins in driving tissue morphogenesis through clonal analysis and RNAi.
C2. Identifying a Role for Musashi-1 in Regulation of Tissue Homeostasis and Organ Size
Thelma T. Chiremba, Liang Xu and Kristi L. Neufeld
Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
The coordinated balance between cell proliferation, differentiation, and apoptosis is crucial for normal development and maintenance of tissue homeostasis. Tumor formation or organ degeneration can initiate when the control mechanisms governing tissue homeostasis are compromised. Thus, the identification and understanding of critical regulatory proteins can illuminate new cancer therapeutic targets. One such potential target, the RNA-binding protein Musashi-1 (Msi-1) is upregulated in most colorectal cancers. Msi-1 binds to the 3’-untranslated region (3’-UTR) of specific mRNAs and inhibits translation. We previously identified the critical colon tumor suppressor Adenomatous polyposis coli (APC) as a Msi-1 target and showed that Msi-1 blocks APC translation. The most well characterized role of APC is as an antagonist of the Wnt signaling pathway. Wnt signaling promotes epithelial cell proliferation in intestinal tissue and we identified Msi-1 as a Wnt target gene. Other known Msi-1 targets, including p21 and Numb, play essential roles in regulating proliferation, differentiation, apoptosis, and cell cycle progression. Our identification of a double-negative feedback loop between APC and Msi-1 led us to hypothesize that this relationship is critical for maintaining a balance of intestinal epithelial cell proliferation and differentiation. To characterize the relationship between APC and Msi-1 in vivo, we recently generated an inducible mouse model that overexpresses Msi-1 in all tissues upon tamoxifen administration. Here we provide evidence that the ubiquitous overexpression of Msi-1 results in delayed overall body growth and premature death. These Msi-1 transgenic mice fail to maintain normal body to organ proportions, with particular organs either too small or too large when compared to wild-type mice. Our findings thus far implicate Msi-1 in regulation of organ size during early mammalian development. Current studies are aimed at identifying and characterizing mechanisms through which Msi-1 contributes to the observed phenotypes.
C3. Contribution of Wnt Signaling Pathway to Asymmetric Stem Cell Division in Colon Crypts
Rita-Marie T. McFadden1, Taybor W. Parker1, Victoria A. Vande Griend1 and Kristi L. Neufeld1,2
1Department of Molecular Biosciences, The University of Kansas, Lawrence, KS, U.S.A.; 2Cancer Biology Program, The University of Kansas Cancer Center, Kansas City, KS, U.S.A.
Background: The ability of a stem cell to renew itself, as well as to create a daughter cell that differentiates to a specialized cell is critical for tissue homeostasis. Loss of this asymmetric cell division (ACD) can lead to cancer. Over 80% of colonic tumors have mutations in the Adenomatous Polyposis Coli (APC) gene. APC protein is necessary for a Wnt-inhibited beta-catenin destruction complex (BCD). Our research demonstrates that components of the BCD may contribute to asymmetric cell division.
Methods: We exposed rat intestinal epithelial cells grown in culture to Wnt3a-ligated beads to provide a localized Wnt signal. Cells were stained with antibodies specific to BCD components Adenomatous Plyposis Coli (Apc), Gluycogen synthase kinase 3 beta (GSK-3β), Axin, and Casein kinase 1 alpha (CK1-α) as well as to Numb. We evaluated ACD in colon tissues from mice with a mutation in the Apc gene, resulting in compromised nuclear APC localization (ApcmNLS/mNLS) as well as Apc+/+ littermate controls. Longitudinally Oriented Basal Asymmetry (LOBA) was assessed to identify specific cell curbing orientation characteristic of ACD. Stem cells were identified using in situ RNA staining for Lgr5.
Results: Compared to their Apc+/+ littermates, ApcmNLS/mNLS mice have fewer Lgr5-expressing cells in the crypts of the proximal colon, suggesting fewer Lgr5+ stem cells. We also observed a pattern of lower LOBA values, suggesting less asymmetric cell division in ApcmNLS/mNLS, compared to Apc+/+ littermate control mice. BDC components APC, GSK-3β, Axin, and CK1-α showed varying degrees of Wnt-bead associated asymmetric localization in cultured intestinal epithelial cells.
Conclusions: BCD components undergo ACD in response to Wnt signaling and mice with Apc-mNLS protein display alterations in stem cell numbers and features of ACD. Together, these results reveal a new molecular mechanism underlying control of stem cell division which potentially contributes to colon cancer.
C4. Mesenchymal Stem Cell Secretome-Rich Hydrogels for Cardiovascular Regeneration
Renae Waters1, Settimio Pacelli1, Ryan Maloney1, Rafeeq P.H. Ahmed2, and Arghya Paul*1
1Department of Chemical and Petroleum Engineering, Bioengineering Graduate Program, University of Kansas, Lawrence, KS, USA; 1Department of Pathology, University of Cincinnati, Cincinnati, OH, USA
Introduction: Stem cell therapy provides a promising alternative to heart transplantation for end-stage patients post myocardial infarction due to its potential to regenerate damaged myocardium. Recent evidence indicates that the dominant mechanism behind this regeneration is the secretion of a broad repertoire of paracrine factors (growth factors, chemokines and exosomes) secreted by the stem cells, known as secretome . Here, we have (i) used a microfluidic device to efficiently bio-manufacture cell secretome from 3D stem cell aggregates, (ii) demonstrated the ability of a nanocomposite hydrogel to modulate the release of key secretome growth factors and (iii) proven the proangiogenic and cardioprotective properties of this secretome loaded nanocomposite hydrogel system.
Experiments and Results: Initially, a soft lithography technique was used to fabricate a microfluidic device consisting of deep concave microwell arrays. When injected into this device, bone marrow derived human mesenchymal stem cell (hMSC) suspensions formed uniformly sized aggregates (~220μm diameter) within 2 days. After 3 days of incubation hMSC secretome was collected and the therapeutic growth factor (VEGF, FGF2, Angiogenin, and BMP-2) concentrations present were found to be 4.5-9 times greater when compared to 2D culture.
As a next step, hMSC secretome was encapsulated in a chemically cross-linked nanocomposite hydrogel delivery system comprised of silicate nanoplatelets and gelatin methacrylate (GelMA). In vitro studies confirmed that the hydrogel system was able to modulate the release of bioactive hMSC growth factors over a 15 day time period. In addition, the proangiogenic ability of the secretome loaded hydrogel was confirmed by the enhanced proliferation of human endothelial cells (HUVEC). Lastly, cardiomyocytes seeded on the secretome loaded hydrogel were subjected to hypoxic (1% O2) and serum deprived (0% fetal bovine serum) conditions for 24 hours in order to mimic cell apoptosis present in the post-infarct myocardium. A significant decrease in the percentage of apoptotic cells was seen on the secretome loaded hydrogel (15.7±3.4%) in comparison to the hydrogel alone (27.1±4.9%). This data confirms the angiogenic and cardioprotective therapeutic potential of the developed hydrogel in vitro.
Conclusion: This study provides a novel strategy for the use of stem cell derived secretome, as an alternative to traditional cell therapy, for myocardial regeneration therapy. The strategy can also be used for other biomedical applications, such as wound healing and vascular tissue engineering.
 Burdon TJ, Paul A, Noiseux N, Prakash S, Shum-Tim D (2011). Bone marrow stem cell derived paracrine factors for regenerative medicine: current perspectives and therapeutic potential. Bone Marrow Res. 2011:207326.
C5. Targeting metabolic regulation of mutant p53 stability in cancer cells
Alejandro Parrales1 Atul Ranjan1, Swathi V. Iyer1, Scott Weir2, Anuradha Roy3 and Tomoo Iwakuma1
1 Department of Cancer Biology, 2 Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA; 3 High Throughput Screening Laboratory, University of Kansas, Lawrence, KS, USA
More than 50% of human cancers carry mutations in the tumor suppressor p53 gene. Mutations in the p53 gene are mostly missense mutations and result in accumulation of dysfunctional p53 protein in tumors with oncogenic gain-of-function activities, such as metastasis and chemotherapy resistance. Increasing evidence indicates that stabilization of mutant p53 (Mp53) in tumors is crucial for its oncogenic activities, while downregulation of Mp53 reduces oncogenicity of cancer cells. These observations suggest that malignant properties of cancer cells are dependent on the presence of Mp53, thus providing a rationale to identify compounds that deplete mutant p53 with little impact on wild-type p53. Toward this goal, we performed high-throughput screens of approximately 9,000 chemical compounds using a p53-null cell line expressing a fusion protein of p53R175H and luciferase reporter. This screening led us to identify “statins”, a class of cholesterol-lowering medications, as compounds that induced degradation of p53R175H with little effect on wild-type p53 expression. We found that other inhibitors of the mevalonate pathway, such as 6-fluoromevalonate and zoledronic acid, failed to induce p53R175H degradation, while statin-mediated inhibition of HMG-CoA reductase and subsequent reduction in mevalonte-5-phosphate triggered p53R175H degradation. These results suggest that statin’s effect on p53R175H is specific and independent of protein prenylation/lipidation or cholesterol synthesis. Moreover, nuclear export of p53R175H was required for the statin-mediated degradation, which was mediated through an E3 ubiquitin ligase CHIP, but not MDM2. Interestingly, statins induced degradation of mainly conformational p53 mutants with minimal effects on the levels of wild-type p53 and DNA contact mutants. This is the first study demonstrating that Mp53 stability is regulated through a specific process of the mevalonate pathway, thereby providing a novel regulatory mechanism of Mp53 degradation. Our findings suggest that p53 mutation status in tumors may have an impact on efficacy of statins in cancer therapy.
C6. A Genome Wide Screening Identifies Novel Regulators of P53 Activity
Atul Ranjan, Swathi V. Iyer, Alejandro Parrales, Tomoo Iwakuma
Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA
The p53 tumor suppressor protein is a transcriptional factor that plays a key role in regulation of the cell cycle, apoptosis, and other cellular processes. Activation of p53 in cancers carrying wild-type p53 is an attractive and rational strategy to treat tumors. A small molecule Nutlin-3a which is presently in clinical trials competitively binds to the N-terminal region of MDM2, leading to activation of p53 and suppression of tumor growth in vivo. However, it remains unknown exactly how Nutlin-3a activates p53 and suppresses tumor growth and what are the crucial regulators of p53-mediated tumor suppression. Toward this goal, we treated U2OS (wild-type p53) cells infected with a whole genome shRNA lentiviral library with Nutlin-3a for 2 weeks and identified shRNAs in surviving colonies. Four target genes were identified through this screening. Using 2 different shRNAs for each gene, we confirmed that knockdown of APOBEC4, an apolipoprotein B mRNA Editing Enzyme and RUNDC1, a RUN domain containing protein, consistently made U2OS and HCT116 (wild-type p53) resistant to Nutlin-3a treatment. These protein functions are poorly understood. Knockdown of these genes rescued reduced cellular population in S phase of the cell cycle by Nutlin-3a. Quantitative RT-PCR analyses revealed that knockdown of these genes attenuated mRNA upregulation of p21, a p53 downstream target, by Nutlin-3a treatment. Chromatin-immunoprecipitation assays also verified reduced p53 binding to the promoter region of p21 in cells downregulated for these proteins. Currently, dissection of the mechanisms by which APOBEC4 and RUNDC1 inhibit p53 activity induced by Nutlin-3a is underway. Completion of our study will identify novel essential activators or cofactors for p53 activation which may acts as tumor suppressors. Suppression of these protein expressions in cancer cells could be a potential escaping mechanism from cell cycle arrest or cell death in cancer cells treated with Nutlin-3a.
C7. Screening of Novel Compounds as Potential Inhibitors of Hu Antigen R–mRNA binding
Sarena L. Senegal1, Xiaoqing Wu2, Thiwanaka B. Samarakoon1, Liang Xu2, Paul R. Hanson1
1Department of Chemistry, University of Kansas, Lawrence, KS, USA; 2Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
We are interested in screening novel compounds for their ability to disrupt mRNA binding to Hu Antigen R (HuR), an over expressed protein in various cancers. HuR is known to bind to its target mRNA at adenine and uridines-rich elements (AREs) and through this binding, promote tumorigenesis by increasing the stability and translation of its target mRNA which encode proteins implicated in different tumor processes such as cell proliferation, invasion, and metastasis1 to name a few. Previously we have successfully set up fluorescence polarization (FP) as a method for screening various compounds. Using FP we have performed High throughput screening 10,000 compounds and have established 200 of those compounds as initial hits that exhibit high specificity for binding to HuR and therefore effectively disrupts its interaction with its target mRNA.
C8. Genetic Control of Tissue Specific Growth in the Larval Trachea of Drosophila
Erin Suderman, Kayla Wilson and Rob Ward
Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA
In most organisms, different tissues and organs grow at altered rates relative to each other, suggesting underlying growth mechanisms that act tissue specifically. The mechanisms of tissue specific growth are less well understood than those governing the growth of an entire organism. To gain a better understanding of these tissue specific growth mechanisms, our lab has been characterizing mutations that specifically alter the growth of the larval trachea in Drosophila melanogaster. Larval trachea growth is well suited for these studies since the trachea shows allometric growth during the larval stages, can be imaged and measured in living animals and gene expression can be specifically altered in the trachea using breathless-GAL4. Importantly, we and others have identified mutations in genes whose mutant phenotypes suggest that they normally regulate tissue-specific growth in the larval trachea. For example, animals with mutations in uninflatable (uif) and Matrix metalloproteinase 1 (Mmp1) have larval tracheae that are roughly half the relative size of those in wild type animals. Through EMS screens of larval lethal mutations, we have obtained multiple alleles of a tracheal growth mutant showing enhanced relative tracheal growth during the larval stages. Based on whole genome sequencing and tracheal-specific RNAi expression data, we have mapped this mutation to the extracellular glycine gated chloride channel CG11340. Here we will show the identification and characterization of the CG11340 mutants through whole genome sequencing, RNAi recapitulation, trachea measurements and antibody staining.
C9. Suppression of intestinal tumorigenesis in Apc mutant mice by Musashi-1 deletion
Andy R. Wolfe1, Amanda Ernlund1, William McGuinness1, Carl Lehmann1, Kaitlyn Carl1, Nicole Balmaceda1, Kristi L. Neufeld1,2
1Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA; 1Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA
The RNA-binding protein Musashi 1 (Msi-1) regulates target mRNA translation and is implicated in colon tumorigenesis. We previously identified the tumor suppressor Adenomatous Polyposis Coli (Apc), a Wnt pathway antagonist, as a Msi-1 target. Truncation of Apc initiates ~80% of all colorectal cancers and Msi-1 levels are high in colon tumors. In cultured colon cells, Msi-1 blocks Apc translation and Apc indirectly inhibits transcription of Msi-1, a Wnt target gene. Based on these findings, we previously proposed an Apc/Msi-1 double negative feedback loop and also that Msi-1 might make a good anti-cancer therapeutic target. Here, we tested whether the Msi-1 upregulation that results from Apc loss contributes to colorectal tumorigenesis by eliminating Msi-1 with a germline deletion in two Apc-mutant mouse models of intestinal cancer. Both ApcMin and Apc1322T mice showed a dramatic reduction in intestinal polyp number if they lacked Msi-1. This result provides genetic evidence that Msi-1 plays a key role in intestinal tumorigenesis. Moreover, intestinal epithelial cells from Apc+/+ mice lacking Msi-1 displayed more Apc, and less b-catenin protein and decreased Wnt target gene mRNA than wildtype mice. This evidence of altered Apc/Msi-1 double negative feedback in mice lacking Msi-1 provides a possible underlying mechanism for the reduced intestinal polyp numbers seen in these mice. Finally, mice lacking Msi-1 showed defects in intestinal cell proliferation and stem cell populations, implicating these features in the polyp resistant phenotype. Together, these results provide genetic evidence that Msi-1 contributes to intestinal tumorigenesis and validate the pursuit of Msi-1 inhibitors as chemo-prevention/therapeutic agents.