Nucleoside Diphosphate Kinase-3 (NME3) Enhances TLR5-Induced NFκB Activation.

Bacterial flagellin is a potent activator of NFκB signaling, inflammation, and host innate immunity, and recent data indicate that flagellin represents a novel antitumor ligand acting through toll-like receptor 5 (TLR5) and the NFκB pathway to induce host immunity and aid in the clearance of tumor xenografts. To identify innate signaling components of TLR5 responsible for these antitumor effects, a loss-of-function high-throughput screen was employed utilizing carcinoma cells expressing a dynamic NFκB bioluminescent reporter stimulated by Salmonella typhimurium expressing flagellin. A live cell screen of a siRNA library targeting 691 known and predicted human kinases to identify novel tumor cell modulators of TLR5-induced NFκB activation uncovered several interesting positive and negative candidate regulators not previously recognized, including nucleoside diphosphate kinase 3 (NME3), characterized as an enhancer of signaling responses to flagellin. Targeted knockdown and overexpression assays confirmed the regulatory contribution of NME3 to TLR5-mediated NFκB signaling, mechanistically downstream of MyD88. Furthermore, Kaplan-Meier survival analysis showed that NME3 expression correlated highly with TLR5 expression in breast, lung, ovarian, and gastric cancers, and furthermore, high-level expression of NME3 increased overall survival for patients with breast, lung, and ovarian cancer, but the opposite in gastric cancer. Together, these data identify a previously unrecognized proinflammatory role for NME3 in signaling downstream of TLR5 that may potentiate cancer immunotherapies.Implications: Proinflammatory signaling mediated by innate immunity engagement of flagellin-activated TLR5 in tumor cells results in antitumor effects through NME3 kinase, a positive downstream regulator of flagellin-mediated NFκB signaling, enhancing survival for several human cancers. Mol Cancer Res; 16(6); 986-99. ©2018 AACR.

DAPK3 suppresses acini morphogenesis and is required for mouse development.

UNLABELLED: Death-associated protein kinase (DAPK3) is a serine/threonine kinase involved in various signaling pathways important to tissue homeostasis and mammalian biology. Considered to be a putative tumor suppressor, the molecular mechanism by which DAPK3 exerts its suppressive function is not fully understood and the field lacks an appropriate mouse model. To address these gaps, an in vitro three-dimensional tumorigenesis model was used and a constitutive DAPK3-knockout mouse was generated. In the 3D morphogenesis model, loss of DAPK3 through lentiviral-mediated knockdown enlarged acinar size by accelerated acini proliferation and apoptosis while maintaining acini polarity. Depletion of DAPK3 enhanced growth factor-dependent mTOR activation and, furthermore, enlarged DAPK3 acini structures were uniquely sensitive to low doses of rapamycin. Simultaneous knockdown of RAPTOR, a key mTORC1 component, reversed the augmented acinar size in DAPK3-depleted structures indicating an epistatic interaction. Using a validated gene trap strategy to generate a constitutive DAPK3-knockout mouse, it was demonstrated that DAPK3 is vital for early mouse development. The Dapk3 promoter exhibits spatiotemporal activity in developing mice and is actively expressed in normal breast epithelia of adult mice. Importantly, reduction of DAPK3 expression correlates with the development of ductal carcinoma in situ (DCIS) and more aggressive breast cancer as observed in the Oncomine database of clinical breast cancer specimens. IMPLICATIONS: Novel cellular and mouse modeling studies of DAPK3 shed light on its tumor-suppressive mechanisms and provide direct evidence that DAPK3 has relevance in early development.

Illuminating cancer systems with genetically engineered mouse models and coupled luciferase reporters in vivo.

Bioluminescent imaging (BLI) is a powerful noninvasive tool that has dramatically accelerated the in vivo interrogation of cancer systems and longitudinal analysis of mouse models of cancer over the past decade. Various luciferase enzymes have been genetically engineered into mouse models (GEMM) of cancer, which permit investigation of cellular and molecular events associated with oncogenic transcription, posttranslational processing, protein-protein interactions, transformation, and oncogene addiction in live cells and animals. Luciferase-coupled GEMMs ultimately serve as a noninvasive, repetitive, longitudinal, and physiologic means by which cancer systems and therapeutic responses can be investigated accurately within the autochthonous context of a living animal.

Caspase-activated cell-penetrating peptides reveal temporal coupling between endosomal release and apoptosis in an RGC-5 cell model.

Caspase-activatable cell-penetrating peptide (CPP) probes, designed for efficient cell uptake and specificity via cleavable intramolecular quenched-fluorophore strategies, show promise for identifying and imaging retinal ganglion cell apoptosis in vivo. However, initial cell uptake and trafficking events cannot be visualized because the probes are designed to be optically quenched in the intact state. To visualize subcellular activation events in real-time during apoptosis, a new series of matched quenched and nonquenched CPP probes were synthesized. In both native and staurosporine-differentiated RGC-5 cells, probe uptake was time- and concentration-dependent through clathrine-, caveolin-, and pinocytosis-mediated endocytic mechanisms. During apoptosis, KcapTR488, a novel dual fluorophore CPP probe, revealed by multispectral imaging a temporal coupling of endosomal release and effector caspase activation in RGC-5 cells. The novel CPPs described herein provide new tools to study spatial and temporal regulation of endosomal permeability during apoptosis.

A bioluminescent transposon reporter-trap identifies tumor-specific microenvironment-induced promoters in Salmonella for conditional bacterial-based tumor therapy.

UNLABELLED: Salmonella specifically localize to malignant tumors in vivo, a trait potentially exploitable as a delivery system for cancer therapeutics. To characterize mechanisms and genetic responses of Salmonella during interaction with living neoplastic cells, we custom-designed a promoterless transposon reporter containing bacterial luciferase. Analysis of a library containing 7,400 independent Salmonella transposon insertion mutants in coculture with melanoma or colon carcinoma cells identified five bacterial genes specifically activated by cancer cells: adiY, yohJ, STM1787, STM1791, and STM1793. Experiments linked acidic pH, a common characteristic of the tumor microenvironment, to a strong, specific, and reversible stimulus for activation of these Salmonella genes in vitro and in vivo. Indeed, a Salmonella reporter strain encoding a luciferase transgene regulated by the STM1787 promoter, which contains a tusp motif, showed tumor-induced bioluminescence in vivo. Furthermore, Salmonella expressing Shiga toxin from the STM1787 promoter provided potent and selective antitumor activity in vitro and in vivo, showing the potential for a conditional bacterial-based tumor-specific therapeutic. SIGNIFICANCE: Salmonella, which often encounter acidic environments during classical host infection, may co-opt evolutionarily conserved pathways for tumor colonization in response to the acidic tumor microenvironment. We identified specific promoter sequences that provide a platform for targeted Salmonella-based tumor therapy in vivo.

Discriminating Hodgdon Pyrodex(®) and Triple Seven(®) using gas chromatography-mass spectrometry.

Pyrodex(®) and Triple Seven(®) are black powder substitutes that often find use as fillers in improvised explosive devices, such as pipe bombs. These propellants have essentially the same overall appearance and oxidizers, but different fuels. For example, Pyrodex(®) contains sulfur, sodium benzoate, and dicyandiamide (DCDA), whereas Triple Seven(®) lacks sulfur but also contains 3-nitrobenzoic acid. In this method, intact particles and postblast solid residues were reacted with bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane in acetonitrile for 30 min at 60°C. The resultant trimethylsilyl derivatives of the organic fuels were then analyzed by gas chromatography-mass spectrometry. Each derivative was clearly resolved from other components, and high-quality mass spectra were obtained. In addition, characteristic fragments resulting from loss of a methyl radical from the molecular ion (m/z 163 for sulfur, m/z 171 for DCDA, m/z 179 for benzoic acid, and m/z 224 for nitrobenzoic acid) were able to be monitored.