Cell Line T84 Research Articles

Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B.

Hepatitis B virus (HBV) infection is one of the major problems that threatens global health. There have been many studies on HBV, but the relationship between HBV and host factors is largely unexplored and more studies are needed to clarify these interactions. Filamin B is an actin-binding protein that acts as a cytoskeleton protein, and it is involved in cell development and several signaling pathways. In this study, we showed that filamin B interacted with HBV core protein, and the interaction promoted HBV replication. The interaction between filamin B and core protein was observed in HEK 293T, Huh7 and HepG2 cell lines by co-immunoprecipitation and co-localization immnofluoresence. Overexpression of filamin B increased the levels of HBV total RNAs and pre-genome RNA (pgRNA), and improved the secretion level of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). In contrast, filamin B knockdown inhibited HBV replication, decreased the level of HBV total RNAs and pgRNA, and reduced the secretion level of HBsAg and HBeAg. In addition, we found that filamin B and core protein may interact with each other via four blocks of argentine residues at the C-terminus of core protein. In conclusion, we identify filamin B as a novel host factor that can interact with core protein to promote HBV replication in hepatocytes. Our study provides new insights into the relationship between HBV and host factors and may provide new strategies for the treatment of HBV infection.

Improved Total Synthesis of Tubulysins and Design, Synthesis, and Biological Evaluation of New Tubulysins with Highly Potent Cytotoxicities against Cancer Cells as Potential Payloads for Antibody-Drug Conjugates.

Improved, streamlined total syntheses of natural tubulysins such as V (Tb45) and U (Tb46) and pretubulysin D (PTb-D43), and their application to the synthesis of designed tubulysin analogues (Tb44, PTb-D42, PTb-D47-PTb-D49, and Tb50-Tb120), are described. Cytotoxicity evaluation of the synthesized compounds against certain cancer cell lines revealed a number of novel analogues with exceptional potencies [e.g., Tb111: IC50 = 40 pM against MES SA (uterine sarcoma) cell line; IC50 = 6 pM against HEK 293T (human embryonic kidney cancer) cell line; and IC50 = 1.54 nM against MES SA DX (MES SA with marked multidrug resistance) cell line]. These studies led to a set of valuable structure-activity relationships that provide guidance to further molecular design, synthesis, and biological evaluation studies. The extremely potent cytotoxic compounds discovered in these investigations are highly desirable as potential payloads for antibody-drug conjugates and other drug delivery systems for personalized targeted cancer chemotherapies.

The regulatory roles of Notch in osteocyte differentiation via the crosstalk with canonical Wnt pathways during the transition of osteoblasts to osteocytes.

Osteocytes comprise more than 90% of the cells in bone and are differentiated from osteoblasts via an unknown mechanism. Recently, it was shown that Notch signaling plays an important role in osteocyte functions. To gain insights into the mechanisms underlying the functions of Notch in regulating the transition of osteoblasts to osteocytes, we performed a luciferase assay by cloning the proximal E11 and dentin matrix acidic phosphoprotein 1 (DMP1) promotor regions into pGluc-Basic 2 vectors, which were subsequently transfected into the IDG-SW3 (osteocytes), MC3T3 (osteoblasts) and 293T (non-osteoblastic cells) cell lines. Two approaches were used to activate Notch signaling in vitro. One was a Notch1 extracellular antibody-coated cell culture plate, and the other was transfection of a Hairy/Enhancer of Split 1 (Hes1) overexpression vector. The interaction between the Notch and Wnt signaling pathways was probed by assessing the expression of a series of phosphorylated proteins involved in the cascade of both signaling pathways. Our data suggested that Notch signaling regulates E11 expression through Hes1 activity, while Hes1 solely did not initiate the expression of DMP1. The regulatory function of E11 by Hes1 was not observed in the 293T cell line, indicating a cell context-dependent manner of the Notch signaling pathway. Additionally, we found that Notch inhibited Wnt signaling at the late differentiation stage of osteocytes by both directly repressing phosphorylated Akt and preventing the nuclear aggregation of β-catenin. These findings provide profound understandings of Notch's regulatory function in osteocyte differentiation.

A Rare Variant (rs933717) at FBXO31-MAP1LC3B in Chinese Is Associated With Systemic Lupus Erythematosus.

Recent evidence from genetic, cell biology, and animal model studies has suggested a pivotal role of autophagy in mediating systemic lupus erythematosus (SLE). However, the genetic basis has not yet been thoroughly examined. Therefore, the aim of the present study was to identify additional susceptibility variants in autophagy-related genes along with their functional significance. First, we performed a gene family-based genetic association analysis in SLE patients with the use of ImmunoChip arrays, and then we selected the most strongly associated polymorphisms for replication in additional cohorts. To identify regulatory clues, we analyzed publicly available blood expression quantitative trait locus data and Encyclopedia of DNA Elements dataon transcription factor binding sites and cell type-specific differential expression. Functional effects were tested by luciferase reporter assays, electrophoretic mobility shift assays, and differential gene expression assays. In 14,474 samples, we observed that the rare Chinese variant rs933717T was associated with susceptibility to SLE (0.11% in cases versus 0.87% in controls; P = 2.36 × 10-10 , odds ratio 0.13). The rs933717 risk allele C correlated with increased MAP1LC3B expression; increased MAP1LC3B messenger RNA was observed in SLE patients and in lupus-prone mice. In reporter gene constructs, the risk allele increased luciferase activity up to 2.7-3.8-fold in both HEK 293T and Jurkat cell lines, and the binding of HEK 293T and Jurkat cell nuclear extracts to the risk allele was also increased. We observed a likely genetic association between light chain 3B, a widely used marker for autophagy, and susceptibility to SLE.

Development of a Surrogate Neutralization Assay for Norovirus Vaccine Evaluation at the Cellular Level.

Noroviruses (NoVs) are the main pathogens responsible for sporadic and epidemic nonbacterial gastroenteritis, causing an estimated 219,000 deaths annually worldwide. There is no commercially available vaccine for NoVs, due partly to the difficulty in establishing NoV cell culture models. The histo-blood group antigen (HBGA) blocking assay is used extensively to assess the protective potential of candidate vaccine-elicited antibodies, but there is still no widely used cellular evaluation model. In this study, we have established a cell line-based NoV vaccine evaluation model through the construction of human α1,2-fucosyltransferase 2-overexpressing 293T (293T-FUT2) cell lines. The 293T-FUT2 cells stably expressed H type 2 and Lewis y antigens. Virus-like particles (VLPs) of the NoV prototype strain genogroup I.1 (GI.1) and the predominant strains GII.4 and GII.17 could attach to the cell line efficiently in a dose-dependent manner. Importantly, antisera against these NoV VLPs could inhibit the attachment of the VLPs, where the inhibitory effects measured by the attachment inhibition assay correlated significantly with the antibody levels determined by the HBGA blocking assay. Collectively, our attachment inhibition assay could serve as a surrogate neutralization assay for the evaluation of NoV vaccines at the cellular level.

Cell Penetrating Polymers Containing Guanidinium Trigger Apoptosis in Human Hepatocellular Carcinoma Cells unless Conjugated to a Targeting N-Acetyl-Galactosamine Block.

A series of 3-guanidinopropyl methacrylamide (GPMA)-based polymeric gene delivery vehicles were developed via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers have been evaluated for their cellular internalization ability, transfection efficiency, and cytotoxicity. Two homopolymers: P(GPMA20), P(GPMA34), were synthesized to study the effect of guanidium polymer length on delivery efficiency and toxicity. In addition, an N-acetyl-d-galactosamine (GalNAc)-based hydrophilic block was incorporated to produce diblock polymers, which provides a neutral hydrophilic block that sterically protects plasmid-polymer complexes (polyplexes) from colloidal aggregation and aids polyplex targeting to hepatocytes via binding to asialoglycoprotein receptors (ASGPRs). Polyplexes formed with P(GPMAx) (x = 20, 34) homopolymers were shown to be internalized via both energy-dependent and independent pathways, whereas polyplexes formed with block polymers were internalized through endocytosis. Notably, P(GPMAx) polyplexes enter cells very efficiently but are also very toxic to human hepatocellular carcinoma (HepG2) cells and triggered cell apoptosis. In comparison, the presence of a carbohydrate block in the polymer structures reduced the cytotoxicity of the polyplex formulations and increased gene delivery efficiency with HepG2 cells. Transfection efficiency and toxicity studies were also carried out with HEK 293T (human embryonic kidney) cells for comparison. Results showed that polyplexes formed with the P(GPMAx) homopolymers exhibit much higher transfection efficiency and lower toxicity with HEK 293T cells. The presence of the carbohydrate block did not further increase transfection efficiency in comparison to the homopolymers with HEK 293T cells, likely due to the lack of ASGPRs on the HEK 293T cell line. This study revealed that although guanidinium-based polymers have high membrane permeability, their application as plasmid delivery vehicles may be limited by their high cytotoxicity to certain cell types. Thus, the use of cell penetrating structures in polyplex formulations should be used with caution and carefully tailored toward individual cell/tissue types.

MicroRNA‑191‑5p exerts a tumor suppressive role in renal cell carcinoma

Renal cell carcinoma (RCC) is a common tumor of the urinary system. Previously, miR-191-5p has been reported to be associated with various types of cancer; however, its specific functions in RCC have not been investigated to date. In the present study, the expression of miR-191-5p in the 786-O and ACHN cell lines was detected in vitro by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results of RT-qPCR revealed that miR-191-5p was significantly downregulated in the two cell lines compared with the 293T cell line. miR-191-5p was also significantly downregulated in RCC tissue compared with paired normal tissue. In addition, the effects of miR-191-5p on cell proliferation, migration, invasion and apoptosis were examined by CCK-8, MTT, wound scratch, Transwell and flow cytometry assays. Downregulation of miR-191-5p was observed to promote cell proliferation, migration and invasion, as well as to repress the cell apoptosis of 786-O and ACHN cells. Therefore, the current study suggests that miR-191-5p functions as a tumor suppressor in RCC. Further studies are required to uncover the underlying signaling pathway of miR-191-5p and its potential role as a biomarker for early detection and prognosis prediction, and as a therapeutic target of RCC.

Downregulation of microRNA-483-5p Promotes Cell Proliferation and Invasion by Targeting GFRA4 in Hirschsprung's Disease.

Recent studies have suggested the critical roles of miRNAs for disease progression. miRNA-483-5p (miR-483-5p) was previously found to have a relationship with tumor cell behavior, but its biological function in Hirschsprung's disease (HSCR) remains undefined. Thus, we explored the role of miR-483-5p in the pathogenesis of HSCR. Histological changes of colonic tissues were evaluated by hematoxylin and eosin (HE) staining. Quantitative real-time PCR and western blotting were used to determine relative expression levels of miRNA, mRNA, and proteins in 20 HSCR patients and 20 normal colon tissues. In this study, we found that miR-483-5p expression in HSCR tissues was significantly increased and their downregulation promoted cell proliferation, cell cycle progression and invasion and inhibited cell apoptosis in human 293T and SH-SY5Y cell lines by the CCK-8, flow cytometry, and Transwell assay. GNDF family receptor alpha 4 (GFRA4) was confirmed as a downstream target of miR-483-5p by dual-luciferase reporter gene assay and inversely correlated with miR-483-5p expression in cell lines. Taken together, miR-483-5p may play a crucial role in the pathogenesis of HSCR by targeting GFRA4.

MiR-411 functions as a tumor suppressor in renal cell cancer.

Recent studies have revealed that microRNAs (miRNAs) play important roles as oncogenes or tumor suppressors in tumorigenesis and tumor development, by negatively regulating protein expression. A previous study of microarrays identified that miR-411 was down-regulated in renal cell carcinoma (RCC), while few studies investigating the role of miR-411 in the pathogenesis of RCC have been performed. We assessed the miR-411 expression in RCC and paired adjacent normal tissues, as well as in RCC cell lines and a normal renal cell line, by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Furthermore, the effects of miR-411 on RCC and normal renal cell proliferation, apoptosis and migration were determined using MTT assay, CCK-8 assay, flow cytometry and scratch wound assay following restoration of miR-411 with synthetic mimics. Results of qRT-PCR indicated that the expression of miR-411 was down-regulated in RCC tissues and cell lines when compared with adjacent normal tissues and a normal renal cell line. Further, results of CCK-8, MTT, cell scratch and transwell assay showed that over-expression of miR-411 suppressed RCC cell (786-O and ACHN) proliferation and migration. Flow cytometry assay revealed that miR-411 could induce RCC cell apoptosis. However, overexpression of miR-411 had no obvious effect on normal renal cell line 293T. To sum up, miR-411 is significantly down-regulated and plays a role as a tumor suppressor in RCC. Further studies are warranted to determine the mechanisms of miR-411 in RCC pathogenesis and define the target genes of miR-411 in RCC.

Correction of \u03b2-thalassemia mutant by base editor in human embryos

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB −28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB −28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB −28 (A>G) mutation in the patient’s primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

Spirotriazoline oxindoles: A novel chemical scaffold with in vitro anticancer properties

The design and synthesis of a library of twenty-six spirotriazoline oxindoles and their in vitro evaluation as potential anticancer agents is reported. The antiproliferative activity of the synthesized compounds was assessed against four different cancer cell lines (HCT-116 p53(+/+), HCT-116 p53(−/−), MCF-7, and MDA-MB-231). Four spirotriazoline oxindoles showed selectivity against the four cancer cell lines tested over the non-cancer derived HEK 293T cell line. To characterize the molecular mechanisms involved in compound antitumoral activity, two spirotriazoline oxindoles were selected for further studies. Both compounds were able to induce apoptosis and cell cycle arrest at G0/G1 phase and upregulated p53 steady-state levels, while decreasing its main inhibitor MDM2, in HCT-116 cells. Importantly, cytotoxic effects induced by spirotriazoline oxindoles occurred in cancer cells without eliciting cell death in non-malignant CCD-18Co human colon fibroblasts. In addition, four spirotriazoline oxindoles showed selectivity against the triple-negative breast cancer cell line MDA-MB-231 with IC50 values of 3.5–6.7 μM. These results highlight the anticancer potential of spirotriazoline oxindoles, especially when dealing with aggressive and challenging triple-negative breast cancer.

Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor.

Pilocarpine is a prototypical drug used to treat glaucoma and dry mouth and is classified as either a full or partial muscarinic agonist. Here, we report several unexpected results pertaining to its interaction with muscarinic M3 receptor (M3R). We found that pilocarpine was 1000 times less potent in stimulating mouse-eye pupil constriction than muscarinic agonists oxotremorin-M (Oxo-M) or carbachol (CCh), although all three ligands have similar Kd values for M3R. In contrast to CCh or Oxo-M, pilocarpine does not induce Ca2+ mobilization via endogenous M3R in human embryonic kidney cell line 293T (HEK293T) or mouse insulinoma (MIN6) cells. Pilocarpine also fails to stimulate insulin secretion and, instead, antagonizes the insulinotropic effect of Oxo-M and CCh-induced Ca2+ upregulation; however, in HEK293T or Chinese hamster ovary-K1 cells overexpressing M3R, pilocarpine induces Ca2+ transients like those recorded with another cognate G protein-coupled muscarinic receptor, M1R. Stimulation of cells overexpressing M1R or M3R with CCh resulted in a similar reduction in phosphatidylinositol 4,5-bisphosphate (PIP2). In contrast to CCh, pilocarpine stimulated PIP2 hydrolysis only in cells overexpressing M1R but not M3R. Moreover, pilocarpine blocked CCh-stimulated PIP2 hydrolysis in M3R-overexpressing cells, thus, it acted as an antagonist. Pilocarpine activates extracellular regulated kinase 1/2 in MIN6 cells. The stimulatory effect on extracellular regulated kinase (ERK1/2) was blocked by the Src family kinase inhibitor PP2, indicating that the action of pilocarpine on endogenous M3R is biased toward β-arrestin. Taken together, our findings show that pilocarpine can act as either an agonist or antagonist of M3R, depending on the cell type, expression level, and signaling pathway downstream of this receptor.

MiR-608 regulating the expression of ribonucleotide reductase M1 and cytidine deaminase is repressed through induced gemcitabine chemoresistance in pancreatic cancer cells.

Gemcitabine resistance is the main problem in pancreatic adenocarcinoma patients. Hence, we aimed to identify the correlation between expression of RRM1 and CDA as the resistance genes and their predicted targeting miR-608 in the resistant pancreatic cancer cell lines to gemcitabine. Dual luciferase assay was performed to determine whether both RRM1 and CDA are targeted by miR-608 in 293T and pancreatic cancer cell lines. AsPC-1 and MIA PaCa-2 cell lines became gradually resistant to gemcitabine by exposing to the increasing doses of gemcitabine. After RNA and miRNAs extraction and cDNA conversion, the expressions of RRM1, CDA and miR-608 in all cell lines were studied by quantitative PCR. Pre-miR-608 transfection to the cell lines was done by calcium phosphate method. MTT assay was performed for analyzing the chemo sensitivity of different cell lines to gemcitabine. Luciferase assays showed that miR-608 targeted RRM1 and CDA genes in 293T, AsPC-1 and MIA PaCa-2 cell lines. Compared to parental cell line, resistant MIA PaCa-2 and AsPC-1 cells demonstrated increased expression of RRM1 and CDA. On the other hand the expression of miR-608 in resistant MIA PaCa-2 and AsPC-1 cells was lower than parental cells. Furthermore, transfection of MIA PaCa-2 and AsPC-1 cells by miR-608 lead to decreased expression of RRM1 and CDA and lowered viability of the cells in comparison with scrambled microRNA transfected cells. During resistance induction in pancreatic cancer cells, miR-608 which is targeting RRM1 and CDA is downregulated which leads to upregulation of these genes.

Study on the design, synthesis and structure-activity relationships of new thiosemicarbazone compounds as tyrosinase inhibitors

52 Structure-based thiosemicarbazone compounds bearing various substituted-lipophilic part, including substituted-benzaldehyde, substituted-phenylalkan-1-one and their biphenyl-type thiosemicarbazone analogs, were designed, synthesized and evaluated as new tyrosinase inhibitors. The results demonstrated that 22 compounds have potent inhibitory activities against tyrosinase with the IC50 value of lower than 1.0 μM. On the basis of the obtained experimental data, the structure-activity relationships (SARs) were rationally derived. Besides, the inhibition mechanism and the inhibitory kinetics of selected compounds 3d and 6e were investigated, revealing that such type of compounds were belonged to the reversible and competitive tyrosinase inhibitors. To verify the safety of these developed thiosemicarbazone compounds, four randomly selected compounds 3d, 4e, 6a and 9a were also tested in 293T cell line for the evaluation of the cytotoxicity. Interestingly, all these compounds almost did not perform any toxicity to 293T cells even at a high concentration of 1000 μmol/L. Taken together, these results suggested that such compounds could serve as the highly efficient and more safe candidates for the treatment of tyrosinase-related disorders.

Identification of the first cathelicidin gene from skin of Chinese giant salamanders Andrias davidianus with its potent antimicrobial activity

Cathelicidins, as effector molecules, play important roles against infections and represent a crucial component of the innate immune system in vertebrates. They are widely studied in mammals, but little is known in amphibians. In the present study, we report the identification and characterization of a novel cathelicidin from Chinese giant salamander Andrias davidianus, which is the first study in Caudata amphibian. The cDNA sequence encodes a predicted 148-amino-acid polypeptide, which composed of a 20-residue signal peptide, a 94-residue conserved cathelin domain and a 34-residue mature peptide. From the multiple sequence alignments and phylogenetic analysis, AdCath shared conserved structure with other orthologs and clustered with other amphibian peptides. The tissue expression profiles revealed AdCath was highly expressed in skin. To study the function of AdCath gene, the AdCath precursor protein and mature peptide were recombinantly expressed and chemical synthesized respectively. The rAdCath protein could bind to LPS in a dose-dependent manner. When the concentrations of rAdCath protein and mature peptide were up to 22 μg/mL, they showed significantly cytotoxicity to human 293T cell lines. The rAdCath protein and synthetic peptide could exhibit antibacterial activities detected by the minimum inhibitory concentrations assay. From the SEM assay, the synthetic mature peptide could destroy the membrane of bacteria and cause loss of membrane integrity. Collectively, these findings characterized the first cathelicidin from A. davidianus, and highlighted its potential antimicrobial activities, indicating its important roles in the skin immune response against different bacteria.

Fusion of thoracic vertebra with rib

Cathelicidins, as effector molecules, play important roles against infections and represent a crucial component of the innate immune system in vertebrates. They are widely studied in mammals, but little is known in amphibians. In the present study, we report the identification and characterization of a novel cathelicidin from Chinese giant salamander Andrias davidianus, which is the first study in Caudata amphibian. The cDNA sequence encodes a predicted 148-amino-acid polypeptide, which composed of a 20-residue signal peptide, a 94-residue conserved cathelin domain and a 34-residue mature peptide. From the multiple sequence alignments and phylogenetic analysis, AdCath shared conserved structure with other orthologs and clustered with other amphibian peptides. The tissue expression profiles revealed AdCath was highly expressed in skin. To study the function of AdCath gene, the AdCath precursor protein and mature peptide were recombinantly expressed and chemical synthesized respectively. The rAdCath protein could bind to LPS in a dose-dependent manner. When the concentrations of rAdCath protein and mature peptide were up to 22 μg/mL, they showed significantly cytotoxicity to human 293T cell lines. The rAdCath protein and synthetic peptide could exhibit antibacterial activities detected by the minimum inhibitory concentrations assay. From the SEM assay, the synthetic mature peptide could destroy the membrane of bacteria and cause loss of membrane integrity. Collectively, these findings characterized the first cathelicidin from A. davidianus, and highlighted its potential antimicrobial activities, indicating its important roles in the skin immune response against different bacteria.

Abstract 153: Using Enzyme-Catalyzed Proximity Labeling to Define the Interactome of the Insulin-like Growth Factor-1 Receptor

Introduction: Manipulation of I nsulin-like G rowth F actor- 1 R eceptor (IGF1R) signaling has been proposed to be an important therapeutic for the treatment of heart failure and myocardial infarction. However, activation of this receptor has, under certain conditions, been shown to result in deleterious side-effects such as pathological hypertrophy. Development of therapeutic strategies that avoid these side-effects requires a more comprehensive understanding of the proteins involved in the IGF1R signaling axis. To this end, we have made use of the enzyme-catalyzed proximity labeling technique called BioID. Objective: To comprehensively define the interactome of the activated IGF1R. Methods and Results: Briefly, BioID makes use of a biotin protein ligase called BirA* that promiscuously biotinylates neighboring proteins. The protein of interest is fused with BirA* and is then expressed in a particular cell line. Following administration of an excess amount of biotin, BirA* will biotinylate those proteins that are adjacent to or interact with the protein of interest. Interacting proteins are identified by tandem mass spectrometry (MS/MS). We have made a HEK 293T cell line that stably expresses the BirA* biotin ligase fused to the C-terminus of IGF1R. We performed BioID using this cell line (N=3) to identify a set of proteins that bind to the IGF1R following stimulation by IGF1. This set included many known interactors of the IGF1R, such as IRS2 and SHC1, thereby validating this technique. Of the many interesting novel interactors, we chose to pursue validation of sorting nexin 6 (SNX6) as some SNX proteins have been shown to be involved in receptor recycling to the plasma membrane following stimulation. We have found that SNX6 knockdown results in a dramatic diminution of IGF1-mediated ERK phosphorylation (2-fold; N=4; p <0.05). However, there was no effect on IGF1-mediated Akt phosphorylation (N=4). We are currently assessing the role of SNX6 in IGF1R-mediated function and recycling of this receptor. Conclusion: We have demonstrated the ability of the BioID technique to identify a set of proteins that can interact with the activated IGF1R. Moreover, BioID has identified a number of novel interactors that further define IGF1R signaling and function.

In vitro selection of DNA aptamers against renal cell carcinoma using living cell-SELEX

Renal cell carcinoma (RCC) is the most common form of kidney cancer with poor prognosis. Early diagnosis of RCC would significantly improve patient prognosis and quality of life. In this work, we developed new aptamer probes for RCC by using cell-SELEX (systematic evolution of ligands by exponential enrichment) only after 12 rounds of selection, in which a clear cell renal cell carcinoma (ccRCC) cell line 786-O was used as target cell, and embryonic kidney cell line 293T as negative control cell. The selected aptamers were subjected to flow cytometry and laser confocal fluorescence microscopy to evaluate their binding affinity and selectivity. The dissociation constant Kd values of four selected aptamers are all in the nanomolar range. Aptamer W786-1 with the best binding affinity and a Kd value of 9.4 ± 2.0nM was further optimized and its truncated sequence W786-1S showed considerable affinity to 786-O cells. The proteinase and temperature treatment experiment indicated that W786-1 could recognize the target 786-O cells through surface proteins, and remain good binding affinity and excellent selectivity under physiological conditions. Therefore, on the basis of its excellent targeting properties and functional versatility, W786-1 holds great potential to be used as a molecular probe for identifying and targeting RCC.

Abstract TMEM-039: THE ROLE OF SNAIL IN CELL DIFFERENTIATION STATUS: EPITHELIAL-MESENCHYMAL TRANSITION AND CANCER STEM CELLS

Abstract PURPOSE: The epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and become mesenchymal cells with migratory and invasive properties. EMT is essential for numerous developmental processes such as gastrulation, and is involved in the initiation of metastasis for cancer progression. Cancer cells that undergo EMT are enabled to metastasize to form secondary tumors. One characteristic of advanced tumors is a low expression level of let-7, a miRNA known to be vital in both maintenances of cellular differentiation and tumor suppression. There is a gap in the literature concerning the mechanism of let-7 downregulation. Our purpose in this study is to present data that shows Snail, an EMT inducer, directly represses let-7, hence inducing cancer stem cells. EXPERIMENTAL PROCEDURES: Ovarian, pancreatic and breast cancer cell lines treated with TGFβ or EGF, inducers of EMT, were studied in our experiments. Expression of several markers at protein level was visualized with immunofluorescence and quantified with flow cytometry. Gene expression of mesenchymal, epithelial and pluripotency factors and miRNAs was detected by q-RT-PCR. Chromatin immunoprecipitation of Snail was performed in ovarian cancer lines. Let-7i promoter luciferase with and without Snail were co-transfected into OVSAHO and 293T cell lines and followed by luciferase assays for detection of bioluminescence. Snail was knocked down by lentiviral small hairpin RNA. Snail was overexpressed by estrogen receptor fusion protein. Orthotopic xenografts of patient-derived ovarian cancer cells were used to test the effect of Snail knockdown on tumor burden. RESULTS: In selected ovarian cancer lines, levels of Snail expression correlated directly with Nanog, and indirectly with let-7 expression. Snail bound promoters of let-7 in cell lines tested. Luciferase assays demonstrated direct repression of let-7 transcription by Snail. Metastatic burden was significantly reduced in cell line xenografts in which Snail was knocked down. CONCLUSIONS: Let-7 is confirmed to be negatively related to Snail. Our preliminary data also shows Snail directly represses let-7, therefore induces properties of stemness in cancer cells. Cell line and patient-derived data support the relationship between Snail expression, let-7 downregulation, and the induction of cancer stem cells. Snail knockdown appears to disrupt the cancer stem cell state and reduces disease progression. Citation Format: Hanmin Wang, Alyse Huisken-Hill, Evgeny Chirshev, Juli Unternaehrer-Hamm. THE ROLE OF SNAIL IN CELL DIFFERENTIATION STATUS: EPITHELIAL-MESENCHYMAL TRANSITION AND CANCER STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-039.

Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE.

The present study aimed to identify candidate substrates of ubiquitin-specific protease (USP)13 using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). USP13 is a well-characterized member of the USP family, which regulates diverse cellular functions by cleaving ubiquitin from ubiquitinated protein substrates. However, existing studies indicate that USP13 has no detectable hydrolytic activity in vitro. This finding implies that USP13 likely has different substrate specificity. In this study, a USP cleavage assay was performed using two different types of model substrates (glutathione S-transferase-Ub52 and ubiquitin-β-galactosidase) to detect the deubiquitinating enzyme (DUB) activity of USP13. In addition, a proteomic approach was taken by using 2D-DIGE to detect cellular proteins whose expressoin is significantly altered in 293T cell lines following the overexpression of USP13 or its C345S mutant (the catalytically inactive form). The data indicated that USP13 still has no detectable DUB activity in vitro nor does C345S. The results of 2D-DIGE demonstrated that the expression of several proteins increased or decreased significantly in 293T cells following the overexpression of USP13. Mass spec troscopy analysis of gel spots identified 7 proteins, including 4 proteins with an increased expression, namely vinculin, thimet oligopeptidase, cleavage and polyadenylation specific factor 3, and methylosome protein 50, and 3 proteins with a decreased expression, namely adenylosuccinate synthetase, annexin and phosphoglycerate mutase. In addition, in the samples of 293T cell lines after the overexpression of USP13 and USP13 C345S, vinculin exhibited an increased expression, suggesting that it may be a candidate substrate of USP13. However, sufficient follow-up validation studies are required in order to determine whether vinculin protein directly interacts with USP13.