Lipid Nanoparticle-mediated siRNA Transfer Against PCTAIRE1/PCTK1/Cdk16 Inhibits In Vivo Cancer Growth.

PCTAIRE1/CDK16/PCTK1 plays critical roles in cancer cell proliferation and antiapoptosis. To advance our previously published in vitro results with PCTAIRE1 silencing, we examined the in vivo therapeutic potential of this approach by using small interfering RNA (siRNA) encapsulated by lipid nanoparticles. Therapy experiments of PCTAIRE1 siRNA were performed using human HCT116 colorectal cancer cells and human A2058 melanoma cells. A single dose of PCTAIRE1 siRNA-lipid nanoparticles was found to be highly effective in reducing in vivo PCTAIRE1 expression for up to 4 days as assayed by immunoblotting. Therapy experiments were started 4 days after subcutaneous injection of cancer cells. Treatment with PCTAIRE1 siRNA-lipid nanoparticles (0.5 mg/kg RNA, twice a week) reduced tumor volume and weight significantly compared with the scramble-control group. Histopathological analysis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) showed increased apoptosis of tumor cells treated with PCTAIRE1-siRNA. Overall, our results demonstrate that siRNA treatment targeting PCTAIRE1 is effective in vivo, suggesting that PCTAIRE1 siRNA-lipid nanoparticles might be a novel therapeutic approach against cancer cells.

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.

ATG4B (Autophagin-1) phosphorylation modulates autophagy.

Autophagy is a catabolic cellular mechanism for entrapping cellular macromolecules and organelles in intracellular vesicles and degrading their contents by fusion with lysosomes. Important roles for autophagy have been elucidated for cell survival during nutrient insufficiency, eradication of intracellular pathogens, and counteracting aging through clearance of senescent proteins and mitochondria. Autophagic vesicles become decorated with LC3, a protein that mediates their fusion with lysosomes. LC3 is a substrate of the cysteine protease ATG4B (Autophagin-1), where cleavage generates a C-terminal glycine required for LC3 conjugation to lipids in autophagosomes. ATG4B both cleaves pro-LC3 and also hydrolyzes lipids from cleaved LC3. We show here that phosphorylation of ATG4B at Ser-383 and Ser-392 increases its hydrolyase activity as measured using LC3 as a substrate. Reconstituting atg4b(-/-) cells with phosphorylation-deficient ATG4B showed a role of ATG4B phosphorylation in LC3 delipidation and autophagic flux, thus demonstrating that the cellular activity of ATG4B is modulated by phosphorylation. Proteolytic conversion of pro-LC3 to LC3-I was not significantly impacted by ATG4B phosphorylation in cells. Phosphorylation-deficient ATG4B also showed reduced interactions with the lipid-conjugated LC3 but not unconjugated LC3. Taken together, these findings demonstrate a role for Ser-383 and Ser-392 phosphorylation of ATG4B in control of autophagy.

Inhibition of methyltransferases accelerates degradation of cFLIP and sensitizes B-cell lymphoma cells to TRAIL-induced apoptosis.

Non-Hodgkin lymphomas (NHLs) are characterized by specific abnormalities that alter cell cycle regulation, DNA damage response, and apoptotic signaling. It is believed that cancer cells are particularly sensitive to cell death induced by tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL). However, many cancer cells show blocked TRAIL signaling due to up-regulated expression of anti-apoptotic factors, such as cFLIP. This hurdle to TRAIL's tumor cytotoxicity might be overcome by combining TRAIL-based therapy with drugs that reverse blockages of its apoptotic signaling. In this study, we investigated the impact of a pan-methyltransferase inhibitor (3-deazaneplanocin A, or DZNep) on TRAIL-induced apoptosis in aggressive B-cell NHLs: mantle cell, Burkitt, and diffuse large B-cell lymphomas. We characterized TRAIL apoptosis regulation and caspase activation in several NHL-derived cell lines pre-treated with DZNep. We found that DZNep increased cancer cell sensitivity to TRAIL signaling by promoting caspase-8 processing through accelerated cFLIP degradation. No change in cFLIP mRNA level indicated independence of promoter methylation alterations in methyltransferase activity induced by DZNep profoundly affected cFLIP mRNA stability and protein stability. This appears to be in part through increased levels of cFLIP-targeting microRNAs (miR-512-3p and miR-346). However, additional microRNAs and cFLIP-regulating mechanisms appear to be involved in DZNep-mediated enhanced response to extrinsic apoptotic stimuli. The capacity of DZNep to target cFLIP expression on multiple levels underscores DZNep's potential in TRAIL-based therapies for B-cell NHLs.

Myeloperoxidase-dependent lipid peroxidation promotes the oxidative modification of cytosolic proteins in phagocytic neutrophils.

Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.

Novel phosphorylation and ubiquitination sites regulate reactive oxygen species-dependent degradation of anti-apoptotic c-FLIP protein.

The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1ß-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIP(L)) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIP(L) protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIP(L) important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL.