Schlafen 5 as a novel therapeutic target in pancreatic ductal adenocarcinoma.

We provide evidence that a member of the human Schlafen (SLFN) family of proteins, SLFN5, is overexpressed in human pancreatic ductal adenocarcinoma (PDAC). Targeted deletion of SLFN5 results in decreased PDAC cell proliferation and suppresses PDAC tumorigenesis in in vivo PDAC models. Importantly, high expression levels of SLFN5 correlate with worse outcomes in PDAC patients, implicating SLFN5 in the pathophysiology of PDAC that leads to poor outcomes. Our studies establish novel regulatory effects of SLFN5 on cell cycle progression through binding/blocking of the transcriptional repressor E2F7, promoting transcription of key genes that stimulate S phase progression. Together, our studies suggest an essential role for SLFN5 in PDAC and support the potential for developing new therapeutic approaches for the treatment of pancreatic cancer through SLFN5 targeting.

IFN-γ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5.

It is well established that activation of the transcription factor signal transducer and activator of transcription 1 (STAT1) is required for the interferon-γ (IFN-γ)-mediated antiviral response. Here, we found that IFN-γ receptor stimulation also activated Unc-51-like kinase 1 (ULK1), an initiator of Beclin-1-mediated autophagy. Furthermore, the interaction between ULK1 and the mitogen-activated protein kinase kinase kinase MLK3 (mixed lineage kinase 3) was necessary for MLK3 phosphorylation and downstream activation of the kinase ERK5. This autophagy-independent activity of ULK1 promoted the transcription of key antiviral IFN-stimulated genes (ISGs) and was essential for IFN-γ-dependent antiviral effects. These findings define a previously unknown IFN-γ pathway that appears to be a key element of the antiviral response.

Slfn2 Regulates Type I Interferon Responses by Modulating the NF-κB Pathway.

Although members of the Slfn family have been implicated in the regulation of type I interferon (IFN) responses, the mechanisms by which they mediate their effects remain unknown. In the present study, we provide evidence that targeted disruption of the Slfn2 gene leads to increased transcription of IFN-stimulated genes (ISGs) and enhanced type I IFN-mediated antiviral responses. We demonstrate that Slfn2 interacts with protein phosphatase 6 regulatory subunit 1 (PPP6R1), leading to reduced type I IFN-induced activation of nuclear factor kappa B (NF-κB) signaling, resulting in reduced expression of ISGs. Altogether, these data suggest a novel mechanism by which Slfn2 controls ISG expression and provide evidence for a critical role for Slfn2 in the regulation of IFN-mediated biological responses.

A simple, low-cost staining method for rapid-throughput analysis of tumor spheroids.

Tumor spheroids are becoming an important tool for the investigation of cancer stem cell (CSC) function in tumors; thus, low-cost and high-throughput methods for drug screening of tumor spheroids are needed. Using neurospheres as non-adherent three-dimensional (3-D) cultures, we developed a simple, low-cost acridine orange (AO)-based method that allows for rapid analysis of live neurospheres by fluorescence microscopy in a 96-well format. This assay measures the cross-section area of a spheroid, which corresponds to cell viability. Our novel method allows rapid screening of a panel of anti-proliferative drugs to assess inhibitory effects on the growth of cancer stem cells in 3-D cultures.

Differential Regulation of ZEB1 and EMT by MAPK-Interacting Protein Kinases (MNK) and eIF4E in Pancreatic Cancer.

UNLABELLED: Human pancreatic ductal adenocarcinoma (PDAC) tumors are associated with dysregulation of mRNA translation. In this report, it is demonstrated that PDAC cells grown in collagen exhibit increased activation of the MAPK-interacting protein kinases (MNK) that mediate eIF4E phosphorylation. Pharmacologic and genetic targeting of MNKs reverse epithelial-mesenchymal transition (EMT), decrease cell migration, and reduce protein expression of the EMT-regulator ZEB1 without affecting ZEB1 mRNA levels. Paradoxically, targeting eIF4E, the best-characterized effector of MNKs, increases ZEB1 mRNA expression through repression of ZEB1-targeting miRNAs, miR-200c and miR-141. In contrast, targeting the MNK effector hnRNPA1, which can function as a translational repressor, increases ZEB1 protein without increasing ZEB1 mRNA levels. Importantly, treatment with MNK inhibitors blocks growth of chemoresistant PDAC cells in collagen and decreases the number of aldehyde dehydrogenase activity-positive (Aldefluor+) cells. Significantly, MNK inhibitors increase E-cadherin mRNA levels and decrease vimentin mRNA levels in human PDAC organoids without affecting ZEB1 mRNA levels. Importantly, MNK inhibitors also decrease growth of human PDAC organoids. IMPLICATIONS: These results demonstrate differential regulation of ZEB1 and EMT by MNKs and eIF4E, and identify MNKs as potential targets in pancreatic cancer.

Concordance of deregulated mechanisms unveiled in underpowered experiments: PTBP1 knockdown case study.

BACKGROUND: Genome-wide transcriptome profiling generated by microarray and RNA-Seq often provides deregulated genes or pathways applicable only to larger cohort. On the other hand, individualized interpretation of transcriptomes is increasely pursued to improve diagnosis, prognosis, and patient treatment processes. Yet, robust and accurate methods based on a single paired-sample remain an unmet challenge. METHODS: "N-of-1-pathways" translates gene expression data profiles into mechanism-level profiles on single pairs of samples (one p-value per geneset). It relies on three principles: i) statistical universe is a single paired sample, which serves as its own control; ii) statistics can be derived from multiple gene expression measures that share common biological mechanisms assimilated to genesets; iii) semantic similarity metric takes into account inter-mechanisms' relationships to better assess commonality and differences, within and cross study-samples (e.g. patients, cell-lines, tissues, etc.), which helps the interpretation of the underpinning biology. RESULTS: In the context of underpowered experiments, N-of-1-pathways predictions perform better or comparable to those of GSEA and Differentially Expressed Genes enrichment (DEG enrichment), within-and cross-datasets. N-of-1-pathways uncovered concordant PTBP1-dependent mechanisms across datasets (Odds-Ratios≥13, p-values≤1 × 10-5), such as RNA splicing and cell cycle. In addition, it unveils tissue-specific mechanisms of alternatively transcribed PTBP1-dependent genesets. Furthermore, we demonstrate that GSEA and DEG Enrichment preclude accurate analysis on single paired samples. CONCLUSIONS: N-of-1-pathways enables robust and biologically relevant mechanism-level classifiers with small cohorts and one single paired samples that surpasses conventional methods. Further, it identifies unique sample/ patient mechanisms, a requirement for precision medicine.

Impaired hippocampal long-term potentiation in melatonin MT2 receptor-deficient mice.

The pineal product melatonin that acts on specific melatonin receptors has been implicated in pathobiological mechanisms of neuropsychiatric disorders including Alzheimer's disease. We used mice lacking melatonin MT(2) receptors (MT(2) knockouts) to investigate the role of these receptors in synaptic plasticity and learning-dependent behavior. In field CA1 of hippocampal slices from wild-type mice, theta burst stimulation induced robust and stable long-term potentiation that was smaller and decremental in slices from MT(2) knockouts. Tested in an elevated plus-maze on two consecutive days, wild-type mice showed shorter transfer latencies to enter a closed arm on the second day; this experience-dependent behavior did not occur in MT(2) knockouts. These results suggest that MT(2) receptors participate in hippocampal synaptic plasticity and in memory processes.

Drug- and region-specific effects of protracted antidepressant and cocaine treatment on the content of melatonin MT(1) and MT(2) receptor mRNA in the mouse brain.

OBJECTIVES: In the mammalian brain, G protein-coupled MT(1) and MT(2) melatonin receptors may be involved in Alzheimer's pathology, long-term potentiation, depression, and in the behavioral effects of psychoactive drugs. These drugs; e.g. antidepressants and drugs of abuse, are typically used over long periods of time and may alter neuroplasticity and gene expression. We hypothesized that such antidepressant- and cocaine-altered expression of melatonin receptor mRNA may occur in the hippocampus and striatum. METHODOLOGY: Male C3H/HeJ mice were treated with the antidepressants fluoxetine, desipramine, and clomipramine, with the psychostimulant cocaine, and with a vehicle either a single time or once a day for 14 days. Brain samples were collected 24 h after the last injection and the content of MT(1) and MT(2) mRNA was assayed. RESULTS: A single drug injection did not alter the MT(1) and MT(2) mRNA content. In the hippocampus, protracted treatment with antidepressants increased the amount of MT(1) mRNA (with the exception of fluoxetine) but decreased MT(2) mRNA content; cocaine did not produce any alterations. In the striatum, antidepressants produced the opposite effect on MT(1) mRNA content; they decreased it. They did not significantly alter striatal MT(2) mRNA (we observed a nonsignificant trend to a decrease). Cocaine also decreased striatal MT(1) mRNA content without affecting MT(2) mRNA. CONCLUSION: These results suggest that drug- and region-specific alterations of MT(1)/MT(2) mRNA produced by protracted antidepressants and cocaine treatment may alter MT1/MT2 expression and contribute to long-term neuroplastic effects of these drugs.

The regional and cellular expression profile of the melatonin receptor MT1 in the central dopaminergic system.

The physiological effects of pineal melatonin are primarily mediated by melatonin receptors located in the brain and periphery. Even though there are a number of studies demonstrating the regulatory role of melatonin in the development of dopaminergic behaviors, such as psychostimulant-induced diurnal locomotor sensitization or drug seeking, little is known about the contribution of melatonin receptors (i.e., MT1) to this role. Therefore, as a first step in understanding the functional role of melatonin receptors in dopaminergic behaviors, we focused on determining the expression pattern of MT1 receptors in the dopaminergic system of the human and rodent brain. Regional (e.g., nucleus accumbens shell) and cellular (e.g., tyrosine hydroxylase immunopositive cells) expression of MT1 mRNA was characterized by applying the immuno-laser capture microdissection (immuno-LCM) technique coupled with nested RT-PCR. Moreover, employing quantitative Western immunoblotting and RT-PCR, we found that the mouse MT1 receptor expression presents diurnal variations (i.e., low mRNA and high protein levels at night, ZT21). The dopaminergic system-based presence of MT1 receptor proteins was not limited to rodents; we found these receptors in postmortem human brain as well. Further research is needed to understand the regional/cellular functional role of melatonin receptors in the regulation of dopaminergic behaviors, using models such as melatonin receptor knockout mice.

Involvement of the pineal gland in diurnal cocaine reward in mice.

Contribution of circadian mechanisms to the psychostimulant-induced behaviors has been suggested. The pineal gland is important component of circadian mechanisms. Using pinealectomized mice and sham-operated controls, we tested the contribution of pineal gland to the rewarding effects of cocaine in conditioned place preference test. Experiments were performed both during the day and at night. Controls with intact pineal glands demonstrated significant decrease in cocaine-induced conditioned place preference at night compared to daytime, whereas pinealectomized mice did not show any diurnal differences. Circadian mechanisms regulated by the pineal gland thus appear critically involved in cocaine-induced reward.