Francois Natt

The mTOR Inhibitor RAD001 Sensitizes Tumor Cells to DNA-Damaged Induced Apoptosis through Inhibition of p21 Translation

Although DNA damaging agents have revolutionized chemotherapy against solid tumors, a narrow therapeutic window combined with severe side effects has limited their broader use. Here we show that RAD001 (everolimus), a rapamycin derivative, dramatically enhances cisplatin-induced apoptosis in wild-type p53, but not mutant p53 tumor cells. The use of isogenic tumor cell lines expressing either wild-type mTOR cDNA or a mutant that does not bind RAD001 demonstrates that the effects of RAD001 are through inhibition of mTOR function. We further show that RAD001 sensitizes cells to cisplatin by inhibiting p53-induced p21 expression. Unexpectedly, this effect is attributed to a small but significant inhibition of p21 translation combined with its short half-life. These findings provide the molecular rationale for combining DNA damaging agents with RAD001, showing that a general effect on a major anabolic process may dramatically enhance the efficacy of an established drug protocol in the treatment of cancer patients with solid tumors.

Design of a genome-wide siRNA library using an artificial neural network

The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.66) and siRNAs targeting endogenous genes at mRNA and protein levels. Neural networks trained on a complementary 21-nucleotide (nt) guide sequence were superior to those trained on a 19-nt sequence. BIOPREDsi was used in the design of a genome-wide siRNA collection with two potent siRNAs per gene. When this collection of 50,000 siRNAs was used to identify genes involved in the cellular response to hypoxia, two of the most potent hits were the key hypoxia transcription factors HIF1A and ARNT.

Amino Acids Activate mTOR Complex 1 via Ca2+/CaM Signaling to hVps34

Excess levels of circulating amino acids (AAs) play a causal role in specific human pathologies, including obesity and type 2 diabetes. Moreover, obesity and diabetes are contributing factors in the development of cancer, with recent studies suggesting that this link is mediated in part by AA activation of mammalian target of rapamycin (mTOR) Complex 1. AAs appear to mediate this response through class III phosphatidylinositol 3-kinase (PI3K), or human vacuolar protein sorting 34 (hVps34), rather than through the canonical class I PI3K pathway used by growth factors and hormones. Here we show that AAs induce a rise in intracellular Ca(2+) ([Ca(2+)](i)), which triggers mTOR Complex 1 and hVps34 activation. We demonstrate that the rise in [Ca(2+)](i) increases the direct binding of Ca(2+)/calmodulin (CaM) to an evolutionarily conserved motif in hVps34 that is required for lipid kinase activity and increased mTOR Complex 1 signaling. These findings have important implications regarding the basic signaling mechanisms linking metabolic disorders with cancer progression.

Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rpL11-translation-dependent mechanism of p53 induction

Impaired ribosome biogenesis is attributed to nucleolar disruption and diffusion of a subset of 60S ribosomal proteins, particularly ribosomal protein (rp)L11, into the nucleoplasm, where they inhibit MDM2, leading to p53 induction and cell-cycle arrest. Previously, we demonstrated that deletion of the 40S rpS6 gene in mouse liver prevents hepatocytes from re-entering the cell cycle after partial hepatectomy. Here, we show that this response leads to an increase in p53, which is recapitulated in culture by rpS6-siRNA treatment and rescued by the simultaneous depletion of p53. However, disruption of biogenesis of 40S ribosomes had no effect on nucleolar integrity, although p53 induction was mediated by rpL11, leading to the finding that the cell selectively upregulates the translation of mRNAs with a polypyrimidine tract at their 5′-transcriptional start site (5′-TOP mRNAs), including that encoding rpL11, on impairment of 40S ribosome biogenesis. Increased 5′-TOP mRNA translation takes place despite continued 60S ribosome biogenesis and a decrease in global translation. Thus, in proliferative human disorders involving hypomorphic mutations in 40S ribosomal proteins, specific targeting of rpL11 upregulation would spare other stress pathways that mediate the potential benefits of p53 induction.

Control of the SOST Bone Enhancer by PTH Using MEF2 Transcription Factors

Expression of the osteocyte‐derived bone formation inhibitor sclerostin in adult bone requires a distant enhancer. We show that MEF2 transcription factors control this enhancer and mediate inhibition of sclerostin expression by PTH.

siRNA-mediated knockdown of the serotonin transporter in the adult mouse brain

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressant drugs that increase the extracellular levels of serotonin by blocking the reuptake activity of the serotonin transporter (SERT). Although SSRIs elevate brain serotonergic neurotransmission acutely, their full therapeutic effects involve neurochemical adaptations that emerge following chronic drug administration. The adaptive downregulation of SERT has recently been implicated in the therapeutic response of SSRIs. Interestingly, studies using SERT-knockout mice reveal somewhat paradoxical depression-related effects, probably specific to the downregulation of SERT during early development. However, the behavioral significance of SSRI-mediated downregulation of SERT during adulthood is still unknown. We investigated whether somatic gene manipulation, triggered by infusing short interfering RNA (siRNA) into the ventricular system, would enable the downregulation of SERT in the adult mouse brain. Infusing the SERT-targeting siRNA, for 2 weeks, significantly reduced the mRNA levels of SERT in raphe nuclei. Further, a significant, specific and widespread downregulation of SERT-binding sites was achieved in the brain. In contrast, 2-week infusion of the SSRI, citalopram, produced a widespread downregulation of SERT-binding sites, independent of any alterations at the mRNA level. Irrespective of their mechanisms for downregulating SERT in the brain, infusions of SERT-siRNA or citalopram elicited a similar antidepressant-related behavioral response in the forced swim test. These results signify a role for the downregulation of SERT in mediating the antidepressant action of SSRIs in adults. Further, these data demonstrate that siRNA-induced widespread knockdown of gene expression serves as a powerful tool for assessing the function of endogenous genes in the adult brain.

Genome-wide functional analysis of human cell-cycle regulators

Human cells have evolved complex signaling networks to coordinate the cell cycle. A detailed understanding of the global regulation of this fundamental process requires comprehensive identification of the genes and pathways involved in the various stages of cell-cycle progression. To this end, we report a genome-wide analysis of the human cell cycle, cell size, and proliferation by targeting >95% of the protein-coding genes in the human genome using small interfering RNAs (siRNAs). Analysis of >2 million images, acquired by quantitative fluorescence microscopy, showed that depletion of 1,152 genes strongly affected cell-cycle progression. These genes clustered into eight distinct phenotypic categories based on phase of arrest, nuclear area, and nuclear morphology. Phase-specific networks were built by interrogating knowledge-based and physical interaction databases with identified genes. Genome-wide analysis of cell-cycle regulators revealed a number of kinase, phosphatase, and proteolytic proteins and also suggests that processes thought to regulate G1-S phase progression like receptor-mediated signaling, nutrient status, and translation also play important roles in the regulation of G2/M phase transition. Moreover, 15 genes that are integral to TNF/NF-κB signaling were found to regulate G2/M, a previously unanticipated role for this pathway. These analyses provide systems-level insight into both known and novel genes as well as pathways that regulate cell-cycle progression, a number of which may provide new therapeutic approaches for the treatment of cancer.

mGluR7 facilitates extinction of aversive memories and controls amygdala plasticity

Formation and extinction of aversive memories in the mammalian brain are insufficiently understood at the cellular and molecular levels. Using the novel metabotropic glutamate receptor 7 (mGluR7) agonist AMN082, we demonstrate that mGluR7 activation facilitates the extinction of aversive memories in two different amygdala-dependent tasks. Conversely, mGluR7 knockdown using short interfering RNA attenuated the extinction of learned aversion. mGluR7 activation also blocked the acquisition of Pavlovian fear learning and its electrophysiological correlate long-term potentiation in the amygdala. The finding that mGluR7 critically regulates extinction, in addition to acquisition of aversive memories, demonstrates that this receptor may be relevant for the manifestation and treatment of anxiety disorders.

Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells

BackgroundThe mitotic spindle is a complex mechanical apparatus required for accurate segregation of sister chromosomes during mitosis. We designed a genetic screen using automated microscopy to discover factors essential for mitotic progression. Using a RNA interference library of 49,164 double-stranded RNAs targeting 23,835 human genes, we performed a loss of function screen to look for small interfering RNAs that arrest cells in metaphase.ResultsHere we report the identification of genes that, when suppressed, result in structural defects in the mitotic spindle leading to bent, twisted, monopolar, or multipolar spindles, and cause cell cycle arrest. We further describe a novel analysis methodology for large-scale RNA interference datasets that relies on supervised clustering of these genes based on Gene Ontology, protein families, tissue expression, and protein-protein interactions.ConclusionThis approach was utilized to classify functionally the identified genes in discrete mitotic processes. We confirmed the identity for a subset of these genes and examined more closely their mechanical role in spindle architecture.

Amyloid precursor protein knockdown by siRNA impairs spontaneous alternation in adult mice

The cleavage‐product of amyloid precursor protein (APP) constitutes the core component of plaques found in the brains of Alzheimer’s disease (AD) patients. APP is ubiquitously expressed and its precise physiological functions remain unclear. This protein has been proposed to regulate synaptic function and processes underlying learning and memory. While APP knockout mice show behavioral impairments, these may occur due to early changes during development and/or due to abolition of APP function in adult. To investigate the acute effects of APP knockdown without involving developmental processes, APP expression was reduced using RNA interference in adult mouse brain. Small interfering RNAs (siRNAs) that down‐regulated mouse APP protein levels (APP‐siRNA) were identified using an APP plasmid‐siRNA co‐transfection assay in mouse NIH/3T3 fibroblast cells. Infusion of APP‐siRNAs into the ventricular system for 2 weeks also down‐regulated APP mRNA in mouse brain. Highest knockdown of APP mRNA levels was found in the CA2‐CA3 regions of the hippocampus. Mice treated with the most active APP‐siRNA showed a significant reduction in spontaneous alternation rate in the Y‐maze, without effects on forelimb grip strength or locomotor activity. These data suggest that acute knockdown of APP in adult mouse brain impairs hippocampus‐dependent spatial working memory.