BCL‑3 promotes cyclooxygenase‑2/prostaglandin E2 signalling in colorectal cancer.
First discovered as an oncogene in leukaemia, recent reports highlight an emerging role for the proto‑oncogene BCL‑3 in solid tumours. Importantly, BCL‑3 expression is upregulated in >30% of colorectal cancer cases and is reported to be associated with a poor prognosis. However, the mechanism by which BCL‑3 regulates tumorigenesis in the large intestine is yet to be fully elucidated. In the present study, it was shown for the first time that knocking down BCL‑3 expression suppressed cyclooxygenase‑2 (COX‑2)/prostaglandin E2 (PGE2) signalling in colorectal cancer cells, a pathway known to drive several of the hallmarks of cancer. RNAi‑mediated suppression of BCL‑3 expression decreased COX‑2 expression in colorectal cancer cells both at the mRNA and protein level. This reduction in COX‑2 expression resulted in a significant and functional reduction (30‑50%) in the quantity of pro‑tumorigenic PGE2 produced by the cancer cells, as shown by enzyme linked immunoassays and medium exchange experiments. In addition, inhibition of BCL‑3 expression also significantly suppressed cytokine‑induced (TNF‑α or IL‑1β) COX‑2 expression. Taken together, the results of the present study identified a novel role for BCL‑3 in colorectal cancer and suggested that expression of BCL‑3 may be a key determinant in the COX‑2‑meditated response to inflammatory cytokines in colorectal tumour cells. These results suggest that targeting BCL‑3 to suppress PGE2 synthesis may represent an alternative or complementary approach to using non‑steroidal anti‑inflammatory drugs [(NSAIDs), which inhibit cyclooxygenase activity and suppress the conversion of arachidonic acid to prostaglandin], for prevention and/or recurrence in PGE2‑driven tumorigenesis.
High-throughput gene screen reveals modulators of nuclear shape.
Irregular nuclear shapes characterized by blebs, lobules, micronuclei, or invaginations are hallmarks of many cancers and human pathologies. Despite the correlation between abnormal nuclear shape and human pathologies, the mechanism by which the cancer nucleus becomes misshapen is not fully understood. Motivated by recent evidence that modifying chromatin condensation can change nuclear morphology, we conducted a high-throughput RNAi screen to identify epigenetic regulators that are required to maintain normal nuclear shape in human breast epithelial MCF-10A cells. We silenced 607 genes in parallel using an epigenetics siRNA library and used an unbiased Fourier analysis approach to quantify nuclear contour irregularity from fluorescent images captured on a high-content microscope. Using this quantitative approach, which we validated with confocal microscopy, we significantly expand the list of epigenetic regulators that impact nuclear morphology.
Identification and comparison of the porcine H1, U6, and 7SK RNA polymerase III promoters for short hairpin RNA expression.
RNA polymerase III is an essential enzyme in eukaryotes for synthesis of tRNA, 5S rRNA, and other small nuclear and cytoplasmic RNAs. Thus, RNA polymerase III promoters are often used in small hairpin RNA (shRNA) expression. In this study, the porcine H1, U6, and 7SK RNA polymerase III type promoters were cloned into a pcDNA3.1( +) expression vector containing a shRNA sequence targeting enhanced green fluorescent protein (EGFP). PK and DF-1 cells were cotransfected with the construction of recombinant interference expression vector and the EGFP expression vector, pEGFP-N1.
The average fluorescence intensity of EGFP in transfected cells was measured by fluorescence microscopy and flow cytometry. Real-time PCR was used to detect expressed shRNAs and the relative expression of EGFP, to confirm the activity of the promoters. The results showed that the activity of porcine 7SK promoter is stronger than the U6 promoter, which is in turn stronger than porcine H1. While the high levels of expression of the U6 and 7SK promoters saturate the shRNAs level in the host cell, which can cause cytotoxicity and tissue damage. Therefore, porcine H1 promoter is effective for expression of shRNA, and may be an excellent tool to knockdown gene expression in pigs for functional genomics studies. The results also lay a foundation for the development of porcine RNAi technology and genetically modified porcine research.
Amblyomma americanum serpin 41 (AAS41) inhibits inflammation by targeting chymase and chymotrypsin.
Ticks inject serine protease inhibitors (serpins) into their feeding sites to evade serine protease-mediated host defenses against tick-feeding. This study describes two highly identitical (97%) but functionally different Amblyomma americanum tick saliva serpins (AAS41 and 46) that are secreted at the inception of tick-feeding. We show that AAS41, which encodes a leucine at the P1 site inhibits inflammation system proteases: chymase (SI = 3.23, Ka = 5.6 ± 3.7X103M-1 s-1) and α-chymotrypsin (SI = 3.18, Ka = 1.6 ± 4.1X104M-1 s-1), while AAS46, which encodes threonine has no inhibitory activity. Similary, rAAS41 inhibits rMCP-1 purified from rat peritonuem derived mast cells. Consistently, rAAS41 inhibits chymase-mediated inflammation induced by compound 48/80 in rat paw edema and vascular permeability models.
Native AAS41/46 proteins are among tick saliva immunogens that provoke anti-tick immunity in repeatedly infested animals as revealed by specific reactivity with tick immune sera. Of significance, native AAS41/46 play critical tick-feeding functions in that RNAi-mediated silencing caused ticks to ingest significantly less blood. Importantly, monospecific antibodies to rAAS41 blocked inhibitory functions of rAAS41, suggesting potential for design of vaccine antigens that provokes immunity to neutralize functions of this protein at the tick-feeding site. We discuss our findings with reference to tick-feeding physiology and discovery of effective tick vaccine antigens.
Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover.
The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. Here we show that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) and CryAB. NUAK biochemically and genetically interacts with Drosophila Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. We further demonstrate that Fil is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, our results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases.