Targeting netrin-3 in small cell lung cancer and neuroblastoma.

The navigation cue netrin-1 is well-documented for its key role in cancer development and represents a promising therapeutic target currently under clinical investigation. Phase 1 and 2 clinical trials are ongoing with NP137, a humanized monoclonal antibody against netrin-1. Interestingly, the epitope recognized by NP137 in netrin-1 shares 90% homology with its counterpart in netrin-3, the closest member to netrin-1 in humans, for which little is known in the field of cancer. Here, we unveiled that netrin-3 appears to be expressed specifically in human neuroblastoma (NB) and small cell lung cancer (SCLC), two subtypes of neuroectodermal/neuroendocrine lineages. Netrin-3 and netrin-1 expression are mutually exclusive, and the former is driven by the MYCN oncogene in NB, and the ASCL-1 or NeuroD1 transcription factors in SCLC. Netrin-3 expression is correlated with disease stage, aggressiveness, and overall survival in NB. Mechanistically, we confirmed the high affinity of netrin-3 for netrin-1 receptors and we demonstrated that netrin-3 genetic silencing or interference using NP137, delayed tumor engraftment, and reduced tumor growth in animal models. Altogether, these data support the targeting of netrin-3 in NB and SCLC.

Plasma biomarker for detection of early stage pancreatic cancer and risk factors for pancreatic malignancy using antibodies for apolipoprotein-AII isoforms.

We recently reported that circulating apolipoprotein AII (apoAII) isoforms apoAII-ATQ/AT (C-terminal truncations of the apoAII homo-dimer) decline significantly in pancreatic cancer and thus might serve as plasma biomarkers for the early detection of this disease. We report here the development of novel enzyme-linked immunosorbent assays (ELISAs) for measurement of apoAII-ATQ/AT and their clinical applicability for early detection of pancreatic cancer. Plasma and serum concentrations of apoAII-ATQ/AT were measured in three independent cohorts, which comprised healthy control subjects and patients with pancreatic cancer and gastroenterologic diseases (n = 1156). These cohorts included 151 cases of stage I/II pancreatic cancer. ApoAII-ATQ/AT not only distinguished the early stages of pancreatic cancer from healthy controls but also identified patients at high risk for pancreatic malignancy. AUC values of apoAII-ATQ/AT to detect early stage pancreatic cancer were higher than those of CA19-9 in all independent cohorts. ApoAII-ATQ/AT is a potential biomarker for screening patients for the early stage of pancreatic cancer and identifying patients at risk for pancreatic malignancy (161 words).

ROS production and apoptosis induction by formation of Gts1p-mediated protein aggregates.

GTS1 of Saccharomyces cerevisiae is a pleiotropic gene. Its induction leads to a variety of biological phenomena represented by cell aggregation. The C-terminal polyglutamine sequence in Gts1p is indispensable for its pleiotropy and nuclear localization. This sequence is often observed in polyglutamine diseases, such as Huntington disease, and is believed to induce protein aggregation, leading to cell death. In this study, protein aggregates were formed in a polyglutamine-dependent manner in cells inducing GTS1, and heat-shock protein family, translation elongation factor, and mitochondrial proteins were trapped in Gts1p-mediated protein aggregates. Moreover, the polyglutamine sequence of Gts1p was indispensable to the induction of reactive oxygen species (ROS) production and apoptosis. Deletion of the genes encoding Por1p and Yhb1p altered the profiles of ROS production and apoptosis caused by GTS1 induction, suggesting that the trapping of these proteins in Gts1p-mediated protein aggregates inhibits the intrinsic functions of these proteins.

GTS1 induction causes derepression of Tup1-Cyc8-repressing genes and chromatin remodeling through the interaction of Gts1p with Cyc8p.

GTS1 in yeast Saccharomyces cerevisiae is a pleiotropic gene, the expression of which affects a wide range of biological phenomena. A genome-wide transcriptional analysis identified genes upregulated in response to GTS1 induction, most of which were found to be regulated by the Tup1-Cyc8 co-repressor. Among the upregulated genes, FLO1 was indispensable for cell aggregation, one of the pleiotropic effects of GTS1. Deletion of genes encoding the chromatin remodeling complex SWI/SNF abolished FLO1 upregulation and decreased cell aggregation, although the nuclear localization of Gts1p required for cell aggregation was not affected. GTS1 induction caused chromatin remodeling at the FLO1 promoter in a SWI/SNF-dependent manner on micrococcal nuclease accessibility assay. Cyc8p was detected in Gts1p-mediated protein aggregates by agarose gel electrophoresis, indicating that Gts1p inhibited Cyc8p function. These results suggest that inhibition of the Tup1-Cyc8 complex and subsequent SWI/SNF-dependent chromatin remodeling result in Gts1p-mediated gene derepression.

Inhibition of heat tolerance and nuclear import of Gts1p by Ssa1p and Ssa2p.

The Saccharomyces cerevisiae gene GTS1 is pleiotropic. GTS1 induction produces a variety of biological phenomena represented by heat tolerance. To clarify the interaction partners of Gts1p, tandem affinity purification and immunoprecipitation were performed. Ssa1p and Ssa2p, members of the 70-kDa heat-shock protein family, were identified. Co-expression of SSA1 or SSA2 inhibited Gts1p nuclear import. As compared to the wild type, the SSA1 and SSA2 double-deletion mutant showed enhancement of Gts1p-mediated heat tolerance in the stationary phase, although neither of the single deletions affected heat tolerance, irrespective of GTS1 induction. These results indicate that the heat tolerance function of Gts1p is regulated by Ssa1p and Ssa2p. Furthermore, time-dependent production of Ssa1p and Ssa2p revealed that Gts1p controls the production of Ssa1p and Ssa2p, and that the total amounts of Ssa1p and Ssa2p are important in inhibiting the unique function of Gts1p.

Application of the arming system for the expression of the 380R antigen from red sea bream iridovirus (RSIV) on the surface of yeast cells: a first step for the development of an oral vaccine.

The cell surface is a functional interface between the inside and the outside of the cell. Moreover, cells have systems for anchoring surface specific proteins and for confining surface proteins to particular domains on the cell surface. For use in bioindustrial processes applied to oral vaccination, we consider that cell-surface display systems must be useful and that the yeast Saccharomyces cerevisiae, the most suitable microorganism for practical purposes, is available as a host for genetic engineering because it can be subjected to many genetic manipulations. In particular, the rigid structure of the cell makes the yeast suitable for several of the applications. In this study, we describe the expression of one of the target antigens, 380R, from the red sea bream iridovirus (RSIV), which is one of the most common viral diseases in the cultured marine fish Pagrus major in Japan, using the arming yeast system and aiming at its application for oral vaccination. We first performed the molecular cloning and expression of the 380R antigen from RSIV in Escherichia coli. The nucleotide sequence of the 380R antigen was composed of an open reading frame (ORF) of 1360 bp encoding a protein of 453 residues. To prepare a specific antibody against the 380R antigen, the recombinant protein was overexpressed and purified in E. coli. As a result of indirect immunofluorescence with the specific antibody, we could observe the expression of the 380R antigen on the surface of the yeast cells. Thus, we have successfully prepared the source of an oral vaccine using cell-surface display technology in yeast.