Evidence that the V832M E-cadherin germ-line missense mutation does not influence the affinity of alpha -catenin for the cadherin/catenin complex

Mutations in E-cadherin are associated with a number of diseases, and have been shown to contribute to disease progression. In particular, 50% of hereditary diffuse gastric cancer cases have inactivating mutations in the E-cadherin gene. An interesting mutation near the beta-catenin-binding site on the cytoplasmic domain of E-cadherin (V832M) was recently reported that produces full-length protein, but exhibits decreased binding of alpha -catenin to the cadherin/catenin complex. The study was done by transfecting mutant E-cadherin into Chinese hamster ovary fibroblast cells. Here we show that the previously reported characteristics of this mutation do not apply to human epithelial cells expressing this mutant protein and suggest that the mechanism whereby the V832M mutation in human E-cadherin promotes gastric cancer is not yet understood.     

Syntenin-1-mediated small extracellular vesicles promotes cell growth,migration, and angiogenesis by increasing onco-miRNAs secretion in lung cancer cells

Small extracellular vesicles (sEVs) play a pivotal role in tumor progression by mediating intercellular communication in the tumor microenvironment (TME). Syntenin-1 induces malignant tumor progression in various types of human cancers, including human lung cancer and regulates biogenesis of sEVs. However, the function of syntenin-1-regulated sEVs and miRNAs in sEVs remains to be elucidated. In the present study, we aimed to demonstrate the role of oncogenic Ras/syntenin-1 axis in the release of sEVs and elucidate the function of syntenin-1-mediated miRNAs in sEVs in lung cancer progression. The results revealed that oncogenic Ras promoted the release of sEVs by inducing syntenin-1 expression; disruption of syntenin-1 expression impaired the release of sEVs as well as sEV-mediated cancer cell migration and angiogenesis. Moreover, we identified three miRNAs, namely miR-181a, miR-425-5p, and miR-494-3p, as onco-miRNAs loaded into syntenin-1-dependent sEVs. Remarkably, miR-494-3p was highly abundant in sEVs and its release was triggered by syntenin-1 expression and oncogenic Ras. Ectopic expression of the miR-494-3p mimic enhanced the migration and proliferation of lung cancer cells as well as tube formation in endothelial cells; however, the miR-494-3p inhibitor blocked sEV-mediated effects by targeting tyrosine-protein phosphatase nonreceptor type 12 (PTPN12), a tumor suppressor. sEVs promoted tumor growth and angiogenesis by downregulating PTPN12 expression; however, the miR-494-3p inhibitor significantly suppressed these effects in vivo, confirming that miR-494-3p acts as a major onco-miRNA loaded into lung cancer cell-derived sEVs. Eventually, the oncogenic Ras/syntenin-1 axis may induce cancer progression by increasing miR-494-3p loading into sEVs in lung cancer cells in the TME.Subject terms: Cancer microenvironment, Non-small-cell lung cancer, Oncogenesis     

Clinical stage drugs targeting inhibitor of apoptosis proteins purge episomal Hepatitis B viral genome in preclinical models

A major unmet clinical need is a therapeutic capable of removing hepatitis B virus (HBV) genome from the liver of infected individuals to reduce their risk of developing liver cancer. A strategy to deliver such a therapy could utilize the ability to target and promote apoptosis of infected hepatocytes. Presently there is no clinically relevant strategy that has been shown to effectively remove persistent episomal covalently closed circular HBV DNA (cccDNA) from the nucleus of hepatocytes. We used linearized single genome length HBV DNA of various genotypes to establish a cccDNA-like reservoir in immunocompetent mice and showed that clinical-stage orally administered drugs that antagonize the function of cellular inhibitor of apoptosis proteins can eliminate HBV replication and episomal HBV genome in the liver. Primary human liver organoid models were used to confirm the clinical relevance of these results. This study underscores a clinically tenable strategy for the potential elimination of chronic HBV reservoirs in patients.Subject terms: Target validation, Hepatitis B, Preclinical research, Translational research     

Evidence for Multiple Acquisition of <Emphasis Type="Italic">Arsenophonus</Emphasis> by Whitefly Species (Sternorrhyncha: Aleyrodidae)

Whiteflies contain primary prokaryotic endosymbionts located within specialized host cells. This endosymbiotic association is the result of a single infection of the host followed by vertical transmission of the endosymbiont to the progeny. Whiteflies may also be associated with other bacteria called secondary (S-) endosymbionts. The nucleotide sequence of the 16S–23S ribosomal DNA from S-endosymbionts of 13 whitefly species was determined. A phylogenetic analysis of these sequences indicated their grouping into two major clusters, one consisting of two S-endosymbionts related to previously described T-type endosymbionts. The second cluster contained the 16S–23S rDNA sequence of the type strain of Arsenophonus nasoniae as well as sequences of S-endosymbionts from 11 whitefly species. This Arsenophonus cluster contained four S-endosymbionts with intervening sequences of 70–184 nucleotides in their 23S rDNAs. The phylogenetic tree of the Arsenophonus cluster differed greatly from the phylogenetic tree of the primary endosymbionts. These results suggest that, unlike the primary endosymbiont, Arsenophonus may infect whiteflies multiple times and may also be horizontally transmitted.     

Sequence Analysis of DNA Fragments from the Genome of the Primary Endosymbiont of the Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

The whitefly Bemisia tabaci contains a primary prokaryotic endosymbiont housed within specialized cells in the body cavity. Two DNA fragments from the endosymbiont, totaling 33.3 kilobases, were cloned and sequenced. In total, 37 genes were detected and included the ribosomal RNA operon and genes for ribosomal RNA proteins. The guanine plus cytosine of the DNA was 30.2 mol%, different from that of endosymbionts of other plant sap-sucking insects.     

Regulation of DNaseY activity by actinin-alpha4 during apoptosis

DNaseY, a Ca(2+)- and Mg(2+)-dependent endonuclease, has been implicated in apoptotic DNA degradation; however, the molecular mechanisms controlling its involvement in this process have not been fully elucidated. We have obtained evidence from yeast two-hybrid screening and coimmunoprecipitation experiments that DNaseY interacted physically with actinin-alpha4 and this interaction significantly enhanced its endonuclease activity. Accordingly, simultaneous overexpression of both proteins in PC12 cells dramatically increased the rate of apoptosis in response to teniposide' VM26. However, overexpression of DNaseY alone neither triggered apoptosis nor facilitated cell death in response to VM26 or serum deprivation. Instead, the overexpression of DNaseY increased the production of single-strand DNA breaks and evoked a profound upregulation of DNA repair pathways. Taken together, our results point to a novel regulatory mechanism of DNaseY activity and offer an explanation for why cells must first cleave key DNA repair and replication proteins before the successful execution of apoptosis.     

PAK is essential for RAS-induced upregulation of cyclin D1 during the G1 to S transition

Oncogenic RAS mutants such as v-Ha-RAS induce cell cycling, in particular the G1 to S transition, by upregulating cyclin D1 and downregulating p27, an inhibitor for cyclin-dependent kinases (CDKs). PI-3 kinase appears to be involved in the regulation of both cyclin D1 and p27. In this report, using two distinct inhibitors specific for PAK1-3 (CEP-1347 and WR-PAK18), we present the first evidence indicating that the PIX/Rac/CDC42-dependent Ser/Thr kinases PAK1-3, acting downstream of PI-3 kinase and upstream of the Raf/MEK/ERKs kinase cascade, is essential for RAS-induced upregulation of cyclin D1, but not downregulation of p27. Since these PAK-inhibitors block selectively the malignant growth of RAS transformants, in which PAK1 is constitutively activated, but not normal cell growth, it is suggested that RAS transformants are addicted to the high levels of PAK1 for their malignant entry to S phase.     

Double chip protein arrays using recombinant single-chain Fv antibody fragments

Protein arrays permit the parallel analysis of many different markers in a small sample volume. However, the problem of cross-reactivity limits the degree of multiplexing in parallel sandwich immunoassays (using monoclonal antibodies (mAbs)), meaning antibodies must be prescreened in order to reduce false positives. In contrast, we use a second chip surface for the local application of detection antibodies, thereby efficiently eliminating antibody cross-reactions. Here, we illustrate the potential advantages of using single-chain Fv fragments rather than mAbs as capture and detection molecules with this double chip technology.