John Fetter

Pro-apoptotic actions of exisulind and CP461 in SW480 colon tumor cells involve β-catenin and cyclin D1 down-regulation

Exisulind and its analogues are inhibitors of cyclic GMP phosphodiesterases (PDEs) that have been shown to activate and induce protein kinase G, resulting in the induction of apoptosis in colon cancer cells. These drugs also reduce beta-catenin protein levels and decrease cyclin D1 mRNA levels in SW480 cells. Herein we report on studies pertaining to exisulind regulation of beta-catenin levels and activity in colon tumor cells. Exisulind and its higher-affinity PDE analogues, (Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)-indenylacetamide hydrochloride (CP461) and (Z)-1H-indene-3-acetamide, 5-fluoro-2-methyl-N-(phenylmethyl)-1-[(3,4,5-trimethoxyphenyl)methylene] (CP248), reduced beta-catenin, including the nuclear beta-catenin in SW480 cells (EC(50) approximately 200 microM, 1 microM, and <1 microM, respectively). The 50% reduction of beta-catenin was seen in 8-14 hr. There was no change in beta-catenin mRNA. Exisulind-induced beta-catenin reduction was blocked by the proteasomal inhibitor MG132 (Z-leu-Leu-Leu-CHO), indicating that the effect of exisulind involved ubiquitin-proteasomal degradation. A consequence of reduced beta-catenin in SW480 cells was that exisulind, CP461, and CP248 caused a concentration- and time-dependent decrease in cyclin D1 levels (EC(50) approximately 300 microM, 1 microM, and <1 microM, respectively) in 4 hr. The effect was via decreased cyclin D1 mRNA levels. Exisulind-induced degradation of beta-catenin was not blocked by the inhibition of caspase-3 activity and/or apoptosis, and some SW480 cells showed a reduction in beta-catenin levels before the appearance of early apoptosis indicators. Expression of the N-terminal 170 amino acid fragment of beta-catenin reduced the effects of beta-catenin degradation, cyclin D1 reduction, and the apoptosis response to exisulind. These results indicate that exisulind-induced beta-catenin degradation precedes the induction of apoptosis and that the down-regulation of inappropriate beta-catenin-activated genes accounts in part for the pro-apoptotic effects of exisulind and CP461 in colon tumor cells.

Domain-Based Biosensor Assay to Screen for Epidermal Growth Factor Receptor Modulators in Live Cells

Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.

Tagging of Genomic STAT3 and STAT1 with Fluorescent Proteins and Insertion of a Luciferase Reporter in the Cyclin D1 Gene Provides a Modified A549 Cell Line to Screen for Selective STAT3 Inhibitors

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic protein that is constitutively activated in numerous cancer cell lines and human cancers. Another STAT family member, STAT1, possesses cancer-inhibitory properties and can promote apoptosis in tumor cells upon activation. To better characterize these important cancer related genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via zinc finger nuclease (ZFN) - mediated homologous recombination in A549 cells that express aberrantly activated STAT3. We inserted the FP transgenes at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. The integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins. When stimulated with IL-6 or IFN-γ, the cells showed robust nuclear translocation of RFP-STAT3 or STAT1-GFP, respectively. Pre-incubation of cells with a known specific STAT3 inhibitor showed that IFN-γ-induced translocation of STAT1-GFP was not impaired. STAT3 activates multiple downstream targets such as genes involved in cell cycle progression - e.g. cyclin D1. To detect changes in expression of endogenous cyclin D1, we used ZFN technology to insert a secreted luciferase reporter behind the cyclin D1 promoter and separated the luciferase and cyclin D1 coding regions by a 2A sequence to induce a translational skip. The luciferase insertion was made in the RFP-STAT3/STAT1-GFP cell line to have all three reporters in a single cell line. Addition of a STAT3 inhibitor led to suppression of cyclin D1 promoter activity and cell growth arrest. The triple-modified cell line provides a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors in live cells while ensuring that the STAT1 pathway is not affected. This approach of reporting endogenous gene activities using ZFN technology could be applied to other cancer targets.

Endogenous gene tagging with fluorescent proteins.

Human genome manipulation has become a powerful tool for understanding the mechanisms of numerous diseases including cancer. Inserting reporter sequences in the desired locations in the genome of a cell can allow monitoring of endogenous activities of disease related genes. Native gene expression and regulation is preserved in these knock-in cells in contrast to cell lines with target overexpression under an exogenous promoter as in the case of transient transfection or stable cell lines with random integration. The fusion proteins created using the modern genome editing tools are expressed at their physiological level and thus are more likely to retain the characteristic expression profile of the endogenous proteins in the cell. Unlike biochemical assays or immunostaining, using a tagged protein under endogenous regulation avoids fixation artifacts and allows detection of the targets activity in live cells. Multiple gene targets could be tagged in a single cell line allowing for the creation of effective cell-based assays for compound screening to discover novel drugs.

Abstract LB-506: Targeted integrations of fluorescent proteins into STAT3 and STAT1 Loci via ZFN mediated homologous recombination in human cancer cell lines provides a suitable and adequate model system for cell-based testing of new candidate drugs for human tumor treatment

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL STAT3 which is a member of the family of signal transducers and activators of transcription is an oncogene and it is constitutively activated in numerous cancer cell lines and human cancers including breast, lung, head and neck, liver, and pancreas. Another STAT family member STAT1 possesses cancer-inhibitory properties and once activated may promote apoptosis in tumor cells. Thus STAT3 has been identified as a promising drug discovery target for many cancers. Ideally the potential inhibitors of STAT3 should be very selective and should not inactivate STAT1. To better address these cancer-research important genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via Zinc-Finger Nuclease (ZFN) mediated homologous recombination in cancer derived cell lines. ZFN technology is a fast and reliable way to manipulate the genome in a targeted fashion. ZFNs are synthetic proteins engineered to bind DNA at a sequence-specific location and create a double strand break (www.compozrzfn.com). The cells natural machinery repairs the break in one of two ways: non-homologous end joining or homologous recombination. Utilizing the homologous recombination pathway that ZFNs induce, we successfully inserted the FP transgenes into the desired target locations - at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. Three cell lines were created, two with the single STAT tagged and a third with both STATs tagged. Integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins in the A549 cell lines. Functional analyses of knock-in cell lines indicated that both STAT3 and STAT1 native gene regulation is conserved resulting in normal levels of protein expression and preservation of protein function. When stimulated with IL-6 or INF-γ, A549 cells showed fast and robust nuclear translocation of RFP-STAT3 or STAT1-GFP respectively. Cell pre-incubation with a known specific STAT3 inhibitors showed that STAT1-GFP, INF-γ-induced translocation was not impaired. Thus, these cell lines provide a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors while ensuring that the STAT1 pathway is not affected. This approach of zfn-tagging endogenous genes could be applied to other cancer targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-506. doi:1538-7445.AM2012-LB-506