LKB1/PEA3/ΔNp63 Pathway Regulates PTGS-2 (COX-2) Transcription in Lung Cancer Cells Upon Cigarette Smoke Exposure

This is the first study to show that cigarette smoking induced the LKB1/PEA 3/ΔNp63-dependent transcriptional regulation of inflammatory molecules, such as COX-2/PTGS-2. Using mainstream smoke extract (MSE) and sidestream smoke extract (SSE) as modeling tools for primary and secondhand smoking, we found that both MSE and SSE downregulated protein levels for LKB1, while upregulated protein levels for PEA 3 and COX-2 in a dose-dependent manner. Using the endogenous ChIP analysis, we further found that the C/EBPβ, NFκB, NF-Y (CHOP), PEA 3 (ETS) and ΔNp63 proteins bound to the specific area (-550 to -130) of the COX-2 promoter, while forming multiple protein complexes in lung cancer cells exposed to MSE and SSE. Our results define a novel link between various transcription factors occupying the COX-2 promoter and cellular response to cigarette smoke exposure bringing a new component, ΔNp63α, showing a critical role for cooperation between various chromatin components in regulation of COX-2 expression and, therefore strengthening the central role of inflammatory process in tumorigenesis of epithelial cells, especially after cigarette smoke exposure (both primary and secondhand)

The effect of tuning cold plasma composition on glioblastoma cell viability

Previous research in cold atmospheric plasma (CAP) and cancer cell interaction has repeatedly proven that the cold plasma induced cell death. It is postulated that the reactive oxygen species (ROS) and reactive nitrogen species (RNS) play a major role in the CAP cancer therapy. In this paper, we seek to determine a mechanism of CAP therapy on glioblastoma cells (U87) through an understanding of the composition of the plasma, including treatment time, voltage, flow-rate and plasma-gas composition. In order to determine the threshold of plasma treatment on U87, normal human astrocytes (E6/E7) were used as the comparison cell line. Our data showed that the 30 sec plasma treatment caused 3-fold cell death in the U87 cells compared to the E6/E7 cells. All the other compositions of cold plasma were performed based on this result: plasma treatment time was maintained at 30 s per well while other plasma characteristics such as voltage, flow rate of source gas, and composition of source gas were changed one at a time to vary the intensity of the reactive species composition in the plasma jet, which may finally have various effect on cells reflected by cell viability. We defined a term “plasma dosage” to summarize the relationship of all the characteristics and cell viability

Perturbation with Intrabodies Reveals That Calpain Cleavage Is Required for Degradation of Huntingtin Exon 1

Background: Proteolytic processing of mutant huntingtin (mHtt), the protein that causes Huntington's disease (HD), is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD. Methodology/Principal Findings: We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1) clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA) 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V_L12.3, turnover of soluble mHDx-1 in living cells is blocked. Conclusions/Significance: These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications

Impaired Delta Np63 Expression is Associated with Poor Tumor Development in Transitional Cell Carcinoma of the Bladder

The oncogenic isoform of the p63 protein, delta Np63 (ΔNp63), plays an important role in the pathogenesis of many epithelial carcinomas, and emerging evidences suggest that ΔNp63 is a promising drug target. However, the functions of ΔNp63 in transitional cell carcinoma of bladder (TCCB) are poorly defined. In this study, a ΔNp63 shRNA expression vector was transfected into TCCB cell line 5637 and cell cycling, cell proliferation and protein expression were assessed by flow cytometry and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-dimethyl tetrazolium bromide (MTT) assay, and immunohistochemistry, respectively. The ΔNp63 shRNA expression vector was also injected into 5637 cell xenograft tumors in nude mice, and tumor size was measured, tumor tissue morphology was assessed by immunohistopathology and transmission electron microscopy. In the in vitro study, ΔNp63 shRNA transfection caused successful ΔNp63 gene silencing and resulted in significant arrest of cell cycling and cellular proliferation (p<0.05) as well as cyclin D1 expression. In the nude mouse xenograft model, ΔNp63 shRNA greatly inhibited tumor growth, induced tumor cell apoptosis (p<0.05) and resulted in cyclin D1 downregulation. Our data suggest that ΔNp63 may play an oncogenic role in TCCB progression through promoting cell survival and proliferation. Intratumoral administration of ΔNp63-specific shRNA suppressed tumor ΔNp63 expression and cellular proliferation while promoted tumor cellular apoptosis, and therefore inhibited tumor growth and improved survival of xenograft-bearing mice, which was not accompanied by significant signs of systemic toxicity

Neurofilament Heavy Polypeptide Regulates the Akt-β-Catenin Pathway in Human Esophageal Squamous Cell Carcinoma

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/β-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of β-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/β-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways

Premature death and neurologic abnormalities in transgenic mice expressing a mutant huntingtin exon-2 fragment

Huntington's disease (HD) is a fatal neurodegenerative disease characterized pathologically by aggregates composed of N-terminal fragments of the mutant form of the protein huntingtin (htt). The role of these N-terminal fragments in disease pathogenesis has been questioned based in part on studies in transgenic mice. In one important example, mice that express an N-terminal fragment of mutant htt terminating at the C-terminus of exon 2 (termed the Shortstop mouse) were reported to develop robust inclusion pathology without developing phenotypic abnormalities seen in the R6/2 or N171-82Q models of HD, which are also based on expression of mutant N-terminal htt fragments. To further explore the capacity of mutant exon-2 htt fragments to produce neurologic abnormalities (N-terminal 118 amino acids; N118), we generated transgenic mice expressing cDNA that encodes htt N118-82Q with the mouse prion promoter vector. In mice generated in this manner, we demonstrate robust inclusion pathology accompanied by early death and failure to gain weight. These phenotypes are the most robust abnormalities identified in the R6/2 and N171-82Q models. We conclude that the lack of an overt phenotype in the initial Shortstop mice cannot be completely explained by the properties of mutant htt N118 fragments

Huntingtin-mediated axonal transport requires arginine methylation by PRMT6

The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington's disease (HD), we study vesicle-associated HTT and find that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues—very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mutant HTT (mHTT) toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Arginine methylation thus regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD.Telethon-Italy and Autonomous Province of Trento (TCP12013 to M.P.); Association Française contre les Myopathies (AFM-22221 to M.P. and M.B.); PRIN-MUR (2017F2A2C5 to M.P.); National Institutes of Health (1R21NS111768-01 to M.P. and U.B.P.); PROGRAM RARE DISEASES CNCCS-Scarl-Pomezia (M.P.); FONDAZIONE AIRC-Italy (24423 to M.P.); Alzheimer Trento Onlus with the legato Baldrachi (M.B.); the Agence Nationale de la Recherche (ANR-15-JPWG-0003-05 JPND CIRCPROT and ANR-18-CE16-0009-01 AXYON to F.S.) and the Spanish Ministry of Science, Innovation and Universities (RTI2018-096322-B-I00 MCIU/AEI/FEDER-UE to J.J.L.

Huntingtin-mediated axonal transport requires arginine methylation by PRMT6

The huntingtin (HTT) protein transports various organelles, including vesicles containing neurotrophic factors, from embryonic development throughout life. To better understand how HTT mediates axonal transport and why this function is disrupted in Huntington's disease (HD), we study vesicle-associated HTT and find that it is dimethylated at a highly conserved arginine residue (R118) by the protein arginine methyltransferase 6 (PRMT6). Without R118 methylation, HTT associates less with vesicles, anterograde trafficking is diminished, and neuronal death ensues—very similar to what occurs in HD. Inhibiting PRMT6 in HD cells and neurons exacerbates mutant HTT (mHTT) toxicity and impairs axonal trafficking, whereas overexpressing PRMT6 restores axonal transport and neuronal viability, except in the presence of a methylation-defective variant of mHTT. In HD flies, overexpressing PRMT6 rescues axonal defects and eclosion. Arginine methylation thus regulates HTT-mediated vesicular transport along the axon, and increasing HTT methylation could be of therapeutic interest for HD