Search

// Yun-Ju Lai 1 , Jui-Cheng Tsai 1 , Ying-Ting Tseng 1 , Meng-Shih Wu 1 , Wen-Shan Liu 1 , Hoi-Ian Lam 1 , Jei-Hwa Yu 3 , Susan E. Nozell 2 , Etty N. Benveniste 2 1 Department of Life Science, National Taiwan Normal University, Taipei, Taiwan 2 Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, AL, USA 3 Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, AL, USA Correspondence to: Yun-Ju Lai, email: yunjulai@ntnu.edu.tw Etty N. Benveniste, email: tika@uab.edu Keywords: glioblastoma, Rac small G protein, glioblastoma-initiating cells, cancer stem cells, zebrafish xenotransplantation Received: January 21, 2016 Accepted: January 03, 2017 Published: February 01, 2017 ABSTRACT Glioblastoma is the most common and aggressive malignant brain tumor in adults. The existence of glioblastoma stem cells (GSCs) or stem–like cells (stemloids) may account for its invasiveness and high recurrence. Rac proteins belong to the Rho small GTPase subfamily which regulates cell movement, proliferation, and survival. To investigate whether Rac proteins can serve as therapeutic targets for glioblastoma, especially for GSCs or stemloids, we examined the potential roles of Rac1, Rac2 and Rac3 on the properties of tumorspheres derived from glioblastoma cell lines. Tumorspheres are thought to be glioblastoma stem-like cells. We showed that Rac proteins promote the STAT3 and ERK activation and enhance cell proliferation and colony formation of glioblastoma stem-like cells. Knockdown of Rac proteins reduces the expression of GSC markers, such as CD133 and Sox2. The in vivo effects of Rac proteins in glioblastoma were further studied in zebrafish and in the mouse xenotransplantation model. Knocking-down Rac proteins abolished the angiogenesis effect induced by the injected tumorspheres in zebrafish model. In the CD133 + -U373-tumorsphere xenotransplanted mouse model, suppression of Rac proteins decreased the incidence of tumor formation and inhibited the tumor growth. Moreover, knockdown of Rac proteins reduced the sphere forming efficiency of cells derived from these tumors. In conclusion, not only Rac1 but also Rac2 and 3 are important for glioblastoma tumorigenesis and can serve as the potential therapeutic targets against glioblastoma and its stem-like cells.

Human papillomavirus (HPV) high-risk genotypes such as HPV-16 and HPV-18 cause the majority of anogenital tract carcinomas, including cervical cancer, the second most common malignancy in women worldwide. Currently there are no approved antiviral agents that reduce or eliminate HPV and reverse virus-associated pathology. We synthesized and evaluated several alkoxyalkyl acyclic nucleoside phosphonate diesters and identified octadecyloxyethyl benzyl 9-[(2-phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG) as an active compound which strongly inhibited transient amplification of HPV-11, -16, and -18 origin-containing plasmid DNA in transfected cells at concentrations well below its cytotoxic concentrations. ODE-Bn-PMEG demonstrated increased uptake in human foreskin fibroblast cells and was readily converted in vitro to the active antiviral metabolite, PMEG diphosphate. The P-chiral enantiomers of ODE-Bn-PMEG were obtained and appeared to have equivalent antiviral activities against HPV. ODE-Bn-PMEG is a promising candidate for the local treatment of HPV-16 and HPV-18 and other high-risk types, an important unmet medical need.

In eukaryotic cells, genomic DNA is organized into a chromatin structure, which not only serves as the template for DNA-based nuclear processes, but also as a platform integrating intracellular and extracellular signals. Although much effort has been spent to characterize chromatin modifying/remodeling activities, little is known about cell signaling pathways targeting these chromatin modulators. Here, we report that cyclin-dependent kinase 1 (CDK1) phosphorylates the histone H2A deubiquitinase Ubp-M at serine 552 (S552P), and, importantly, this phosphorylation is required for cell cycle progression. Mass spectrometry analysis confirmed Ubp-M is phosphorylated at serine 552, and in vitro and in vivo assays demonstrated that CDK1/cyclin B kinase is responsible for Ubp-M S552P. Interestingly, Ubp-M S552P is not required for Ubp-M tetramer formation, deubiquitination activity, substrate specificity, or regulation of gene expression. However, Ubp-M S552P is required for cell proliferation and cell cycle G 2/M phase progression. Ubp-M S552P reduces Ubp-M interaction with nuclear export protein CRM1 and facilitates Ubp-M nuclear localization. Therefore, these studies confirm that Ubp-M is phosphorylated at S552 and identify CDK1 as the enzyme responsible for the phosphorylation. Importantly, this study specifically links Ubp-M S552P to cell cycle G 2/M phase progression.

The immune system has routinely been the focus of cancer treatments. One of the strategies that targets the immune system is by use of monoclonal antibodies to specifically target tumor antigens leading to immune activation and tumor destruction. Therefore, many biopharmaceutical companies are interested/invested in antibody development, including the development of next-generation antibodies such as bispecific antibodies. The Development Center for Biotechnology (DCB) in Taiwan has developed a new platform for the generation of immunoglobulin kappa chain-bridged single chain bispecific antibody. Using this platform, DCB constructed a kappa domain-bridged HER2/CD3 bispecific single chain antibody known as the LBscbHER2-CD3. Utilizing a mammalian expression system, the antibody can be easily purified by affinity chromatography via kappa fragment capture. Binding affinity studies have revealed that the purified LBscbHER2-CD3 antibody is able to independently as well as simultaneously bind to HER2+ tumor cells and CD3+ T cells at low nanomolar concentrations. In co-culture experiments, the addition of purified LBscbHER2-CD3 antibody resulted in a significant increase in IL-2 production which is a strong indicator of human T cell activation. In addition, the co-engagement of either IL-2 primed or un-primed human T cells and HER2+ tumor cells by the LBscbHER2-CD3 antibody facilitated the killing of tumor cells from breast, colon and pancreatic cancers. Lastly, when administered in vivo, LBscbHER2-CD3 showed strong tumor growth inhibition in SCID mice xenograft models with implanted HER2+ SW480 cancer cells, HT29 human colon carcinoma cells, and Capan-1 human pancreatic cancer cells. In conclusion, the LBscbHER2-CD3 bispecific antibody has strong affinity for both the cancer cell-expressing receptor and the T cell antigen which leads to cancer cell elimination in vitro and in vivo. These results suggest that LBscbHER2-CD3 may have therapeutic potential for drug development in the treatment of human cancers. Citation Format: Yu Hsun Lo, Jei-Hwa Yu, Yu-Hsen Hsui, Ta-Tung Yuan. Developmental and functional characterization of immunoglobulin kappa chain-bridged single chain bi-specific antibody for targeted antitumor therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B018.

The G protein-coupled lysophosphatidic acid 2 (LPA2) receptor elicits prosurvival responses to prevent and rescue cells from apoptosis. However, G protein-coupled signals are not sufficient for the full protective effect of LPA2. LPA2 differs from other LPA receptor subtypes in the C-terminal tail, where it contains a zinc finger-binding motif for the interactions with LIM domain-containing TRIP6 and proapoptotic Siva-1, and a PDZ-binding motif through which it complexes with the NHERF2 scaffold protein. In this report, we identify a unique CXXC motif of LPA2 responsible for the binding to TRIP6 and Siva-1, and demonstrate that disruption of these macromolecular complexes or knockdown of TRIP6 or NHERF2 expression attenuates LPA2-mediated protection from chemotherapeutic agent-induced apoptosis. In contrast, knockdown of Siva-1 expression enhances this effect. Furthermore, a PDZ-mediated direct interaction between TRIP6 and NHERF2 facilitates their interaction with LPA2. Together, these results suggest that in addition to G protein-activated signals, the cooperation embedded in the LPA2-TRIP6-NHERF2 ternary complex provides a novel ligand-dependent signal amplification mechanism that is required for LPA2-mediated full activation of antiapoptotic signaling.

The human papillomavirus (HPV) origin (ori) of DNA replication shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundred base pairs. The HPV type-11 ori spans 103 bp and contains three palindromic binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 origin binding protein. These sites are separated by 64 bp and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 ori DNA bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies of the dependence of looping on salt, ATP, and DTT using full length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N terminal domain as well as factors which may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.

Human and animal papillomavirus DNA replicates as multicopy nuclear plasmids. Replication requires two viral proteins, the origin-recognition protein E2 and the replicative DNA helicase E1. Using genetic, biochemical, and immunofluorescence assays, we demonstrated that efficient nuclear import of the human papillomavirus (HPV) type 11 E1 protein depends on a codominant bipartite nuclear localization sequence (NLS) and on phosphorylation of the serine residues S89 and S93 by the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase, and c-Jun N-terminal protein kinase. The NLS and the MAPK substrates are located within a 50-amino-acid-long peptide near the amino terminus, previously designated the localization regulatory region (LRR). The downstream NLS overlaps the cyclin-binding motif RRL, which is necessary for phosphorylation by the cyclin-dependent kinases to inactivate a dominant nuclear export sequence, also in the LRR. Alanine mutations of the MAPK substrates significantly impaired nuclear import, whereas phospho-mimetic mutations partially restored nuclear import. We further identified two MAPK docking motifs near the C terminus of E1 that are conserved among E1 proteins of many HPVs and bovine papillomavirus type 1. Mutations of these MAPK docking motifs or addition of specific MAPK inhibitors significantly reduced nuclear import. Interestingly, a fraction of the NLS-minus E1 protein was cotransported with the E2 protein into the nucleus and supported transient viral DNA replication. In contrast, E1 proteins mutated in the MAPK docking motifs were completely inactive in transient replication, an indication that additional properties were adversely affected by those changes.

e13011 Background: Glioblastoma is the grade 4 astrocytoma which is notorious for its highly invasive phenotype, very low survival rate and generally poor responses to conventional therapies. Glioblastoma stem-like cells (GSC), which are usually more resistant to therapeutic treatment, may account for the poor prognosis of this disease. Rac (Ras-related C3 botulinum toxin substrate) is a subfamily of Rho small GTPase which function is regulation of actin cytoskeleton rearrangement. While Rac1 is expressed ubiquitous in different tissues and cells, Rac2 is highly expressed in the mesenchymal subtype of glioblastoma according to the TCGA (the Cancer Genome Atlas) database, and Rac3 is mainly expressed in the brain. Methods: We used Rac proteins overexpressing-glioblastoma cellines derived GSC and Rac proteins specific siRNA harboring-GSC to perform colony formation assay and migration assay. Results: Here we report that Rac proteins overexpressing glioblastoma stem-like cells derived from glioblastoma cell lines have higher proliferation rate and stronger responses to LPA-induced cell migration. Knocking-down their expression by specific siRNA reduces the proliferation and migration of these cells. Instead of Rac1, Rac2 and Rac3 are more effective on promoting proliferation and migration of glioblastoma stem-like cells. Moreover, Rac proteins promote glioblastoma progression is associated with activation of JAK-STAT and ERK pathway. Conclusions: Although Rac1 is the most studied one in the Rac family, and has also been implicated in the progression of different cancers, however, it is homogeneously expressed in all different tissues, and plays important roles in normal cellular functions involving cell movement, such as wound healing, make it not a good candidate for specific drug targeting. According to our results, Rac2 or Rac3 serve as a better potential therapeutic targets for glioblastoma treatment.