Summary DNA demethylating agents have shown clinical anti-tumor efficacy via an unknown mechanism of action. Using a combination of experimental and bioinformatics analysis in colorectal cancer cells, we demonstrate that low-dose 5-AZA-CdR targets colorectal cancer initiating cells (CICs) by inducing viral mimicry. This is associated with induction of dsRNAs derived at least in part from endogenous retroviral elements, activation of the MDA5/MAVS RNA recognition pathway and downstream activation of IRF7. Indeed, disruption of virus recognition pathways, by individually knocking down MDA5, MAVS or IRF7, inhibits the ability of 5-AZA-CdR to target colorectal CICs and significantly decreases 5-AZA-CdR long-term growth effects. Moreover, transfection of dsRNA into CICs can mimic the effects of 5-AZA-CdR. Together; our results represent a major shift in understanding the antitumor mechanisms of DNA-demethylating agents and highlight the MDA5/MAVS/IRF7 pathway as a potentially druggable target against CICs.
SUMMARY DNA methylation in promoters is well known to silence genes and is the presumed therapeutic target of methylation inhibitors. Gene body methylation is positively correlated with expression yet its function is unknown. We show that 5-aza-2'-deoxycytidine treatment not only reactivates genes but decreases the over-expression of genes, many of which are involved in metabolic processes regulated by c-MYC. Down-regulation is caused by DNA demethylation of the gene bodies and restoration of high levels of expression requires remethylation by DNMT3B. Gene body methylation may therefore be an unexpected therapeutic target for DNA methylation inhibitors, resulting in the normalization of gene over-expression induced during carcinogenesis. Our results provide direct evidence for a causal relationship between gene body methylation and transcription.
In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multi-well plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Then, automated image analysis software identifies individual cells and measures ~1,500 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes two weeks; feature extraction and data analysis take an additional 1-2 weeks.
Despite being engineered to avoid renal clearance, many cationic polymer (polycation)-based siRNA nanoparticles that are used for systemic delivery are rapidly eliminated from the circulation. Here, we show that a component of the renal filtration barrier-the glomerular basement membrane (GBM)-can disassemble cationic cyclodextrin-containing polymer (CDP)-based siRNA nanoparticles and, thereby, facilitate their rapid elimination from circulation. Using confocal and electron microscopies, positron emission tomography, and compartment modeling, we demonstrate that siRNA nanoparticles, but not free siRNA, accumulate and disassemble in the GBM. We also confirm that the siRNA nanoparticles do not disassemble in blood plasma in vitro and in vivo. This clearance mechanism may affect any nanoparticles that assemble primarily by electrostatic interactions between cationic delivery components and anionic nucleic acids (or other therapeutic entities). pharmacokinetics | glomerulusA major challenge with the use of small interfering RNA (siRNA) in mammals is their delivery to intracellular locations in specific tissues (1). The two most investigated approaches to siRNA delivery involve the combination of siRNA with cationic lipids (lipoplexes, liposomes, micelles) or cationic polymers (polyplexes) (2). Polymer-based siRNA delivery vehicles can be tuned to be nonimmunogenic, nononcogenic, nontoxic, and targeted (3). A targeted nanoparticle formulation of siRNA (not chemically modified) with a cationic, cyclodextrin-containing polymer (CDP)-based delivery vehicle (clinical version denoted CALAA-01) was shown to accumulate in human tumors and deliver functional siRNA from a systemic, i.v. infusion (4). This first-in-human study demonstrated the clinical potential for cationic polymerbased siRNA delivery systems.Like most cationic polymer-based siRNA delivery systems (5-9), the siRNA/CDP nanoparticle is rapidly eliminated from circulation (shown in mice, monkeys, and humans) (10-12). In fact, polymer complexation often does not extend the circulation time of siRNA. The rapid clearance of these siRNA nanoparticles is puzzling because they are engineered to be above the size cutoff for single-pass clearance via renal filtration (13). In understanding the mechanism behind the rapid clearance of this type of cancer therapeutic, we can efficiently seek ways to increase their circulation time and, thus, enhance their anticancer efficacy (3).We hypothesize that the paradoxical renal clearance of polycation-nucleic acid nanoparticles results from their binding and disassembly by components of the renal filtration barrier. Three key properties of such nanoparticles (diameters between 10 and 100 nm, positive zeta potentials, and electrostatically driven selfassembly) make them susceptible to this mechanism of clearance.The renal filtration barrier, located within the glomerulus of the nephron, consists of three layers that must be traversed to enter the urinary space. These three layers are the glomerular endothelial fenestrations (≈100 n...
DNA methylation patterns are established during embryonic development and faithfully copied through somatic cell divisions. Based on our understanding of DNA methylation and other interrelated epigenetic modifications, a comprehensive view of the epigenetic landscape and cancer epigenome is evolving. The cancer methylome is highly disrupted, making DNA methylation an excellent target for anti-cancer therapies. During the last few decades, an increasing number of drugs targeting DNA methylation have been developed in an effort to increase efficacy, stability and to decrease toxicity. The earliest and the most successful epigenetic drug to date, 5-Azacytidine, is currently recommended as the first-line treatment for high risk myelodysplastic syndromes (MDS) patients. Encouraging results from clinical trials have prompted further efforts to elucidate epigenetic alterations in cancer and subsequently develop new epigenetic therapies. This review delineates the latest cancer epigenetic models, recent discovery of hypomethylation agents and their application in the clinic.
Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 ‘walks’ along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases.DOI: http://dx.doi.org/10.7554/eLife.24487.001
The cellular machine Cdc48 functions in multiple biological pathways by segregating its protein substrates from a variety of stable environments such as organelles or multi-subunit complexes. Despite extensive studies, the mechanism of Cdc48 has remained obscure, and its reported structures are inconsistent with models of substrate translocation proposed for other AAA+ ATPases (adenosine triphosphatases). Here, we report a 3.7-angstrom–resolution structure of Cdc48 in complex with an adaptor protein and a native substrate. Cdc48 engages substrate by adopting a helical configuration of substrate-binding residues that extends through the central pore of both of the ATPase rings. These findings indicate a unified hand-over-hand mechanism of protein translocation by Cdc48 and other AAA+ ATPases.
Introduction Brain metastases (BM) occur in up to one third of patients with metastatic breast cancer (MBC), whose incidences and prognoses by breast cancer subtypes in BM have not been well delineated.
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