Suppression of eukaryotic initiation factor 4E prevents chemotherapy-induced alopecia

BACKGROUND: Chemotherapy-induced hair loss (alopecia) (CIA) is one of the most feared side effects of chemotherapy among cancer patients. There is currently no pharmacological approach to minimize CIA, although one strategy that has been proposed involves protecting normal cells from chemotherapy by transiently inducing cell cycle arrest. Proof-of-concept for this approach, known as cyclotherapy, has been demonstrated in cell culture settings. METHODS: The eukaryotic initiation factor (eIF) 4E is a cap binding protein that stimulates ribosome recruitment to mRNA templates during the initiation phase of translation. Suppression of eIF4E is known to induce cell cycle arrest. Using a novel inducible and reversible transgenic mouse model that enables RNAi-mediated suppression of eIF4E in vivo, we assessed the consequences of temporal eIF4E suppression on CIA. RESULTS: Our results demonstrate that transient inhibition of eIF4E protects against cyclophosphamide-induced alopecia at the organismal level. At the cellular level, this protection is associated with an accumulation of cells in G1, reduced apoptotic indices, and was phenocopied using small molecule inhibitors targeting the process of translation initiation. CONCLUSIONS: Our data provide a rationale for exploring suppression of translation initiation as an approach to prevent or minimize cyclophosphamide-induced alopecia.1U01 CA168409 - NCI NIH HHS; P01 CA 87497 - NCI NIH HHS; P30 CA008748 - NCI NIH HHS; MOP-106530 - Canadian Institutes of Health Research; P01 CA013106 - NCI NIH HH

Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5' triphosphate (5'ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5'pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5'pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5'pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5'pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5'pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents

Gata3 antagonizes cancer progression in pten-deficient prostates

Loss of the tumor suppressor PTEN is a common occurrence in prostate cancer. This aberration leads to the ectopic activation of the PI3K-Akt pathway, which promotes tumor growth. Here, we show that the transcription factor Gata3 is progressively lost in Pten-deficient mouse prostate tumors as a result of both transcriptional down-regulation and increased proteasomal degradation. To determine the significance of this loss, we used conditional loss- and gain-of-function approaches to manipulate Gata3 expression levels in prostate tumors. Our results show that Gata3 inactivation in Pten-deficient prostates accelerates tumor invasion. Conversely, enforced expression of GATA3 in Pten-deficient tissues markedly delays tumor progression. In Pten-deficient prostatic ducts, enforced GATA3 prevented Akt activation, which correlated with the down-regulation of Pik3cg and Pik3c2a mRNAs, encoding respectively class I and II PI3K subunits. Remarkably, the majority of human prostate tumors similarly show loss of active GATA3 as they progress to the aggressive castrate-resistant stage. In addition, GATA3 expression levels in hormone-sensitive tumors holds predictive value for tumor recurrence. Together, these data establish Gata3 as an important regulator of prostate cancer progression

Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity

The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 59 triphosphate (59ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 59pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. [...] Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 59pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents

5′pppRNA activates innate immunity and protects mice from lethal influenza infection <i>in vivo</i>.

<p>C57Bl/6 mice were injected intravenously with 25 ug of 5′pppRNA in complex with in vivo-jetPEI. (<b>A–B–C</b>) Mice were treated with 5′pppRNA on the day prior to influenza infection (500 PFU; Day −1) and the day of infection (Day 0). Percent survival (<b>A</b>) and percent weight loss (<b>B</b>) were monitored. (<b>C</b>) Lung viral titers were measured by plaque assay at the indicated day post-infection. Error bars represent SEM from six different animals. ND: not detected. (<b>D</b>) Repetitive administration of 5′pppRNA on the indicated days further decreases lung viral titers following infection with 500 PFU of influenza, as determined by plaque assay on Day 3 post-infection. Error bars represent SEM from five different animals. (<b>E</b>) Mice were infected with 50 PFU of influenza on Day 0. 5′pppRNA was administered prophylactically (Day −1, Day 0) or therapeutically (Day 1, Day 2) and lung viral titers were determined on Day 3. Error bars represent SEM from five different animals. (<b>F</b>) WT, TLR3−/−, MAVS−/− mice were treated with 5′pppRNA and serum IFNβ was quantified by ELISA at 6 h. Error bars represent SEM from three different animals. (<b>G</b>) WT and MAVS−/− mice were treated with 5′pppRNA and infected with influenza (500 PFU). Lungs were collected and homogenized on Day 1 and lung viral titers were determined by plaque assay. Error bars represent SEM from four different animals. (<b>H</b>) IFNα/βR−/− mice were either non-treated or treated with 5′pppRNA and infected with influenza (100 PFU). Survival was monitored for 18 days. Statistical analysis was performed by Student's t test (*, p≤0.05; **, p≤0.01; ***, p≤0.001; ns, not statistically significant).</p

5′pppRNA treatment controls influenza-mediated pneumonia.

<p>Mice were treated with 5′pppRNA in complex in vivo-jetPEI on the day prior to influenza infection (Day −1) and the day of infection (Day 0). Lungs were collected on Day 3 and Day 8 post-infection and stained with hematoxylin and eosin (H&E). For each group, a representative picture showing (<b>A</b>) inflammation and tissue damage and (<b>B</b>) the extent of pneumonia is presented. (<b>C</b>) Inflammation, tissue damage and surface area affected by pneumonia were scored by a veterinary pathologist. Grade 1 = minimal; Grade 2 = modest, rare; Grade 3 = moderate, frequent; Grade 4 = severe, extensive.</p

5′pppRNA stimulates an antiviral and inflammatory response in lung epithelial A549 cells.

<p>(<b>A</b>) Schematic representation of VSV-derived 5′pppRNA and gel analysis. The 5′ppp-containing 67-mer RNA oligonucleotide is derived from the untranslated regions (UTRs) of VSV and the product of <i>in vitro</i> transcription runs as a single product degraded by RNase I. (<b>B</b>) 5′pppRNA or a homologous control RNA lacking a 5′-triphosphate end was mixed with Lipofectamine RNAiMax and transfected at different RNA concentrations (0.1–500 ng/ml) into A549 cells. At 8 h post treatment, whole cell extracts (WCEs) were prepared, resolved by SDS-page and analyzed by immunoblotting for IRF3 pSer396, IRF3, ISG56, NOXA, cleaved caspase 3, PARP and β-actin. Results are from a representative experiment; all immunoblots are from the same samples. (<b>C</b>) A549 cells were transfected with 10 ng/ml 5′pppRNA and WCEs were prepared at different times after transfection (0–48 h), subjected to SDS-PAGE and probed with antibodies for IRF3 pSer-396, IRF3, IRF7, STAT1 pTyr-701, STAT1, ISG56, RIG-I, IκBα pSer-32, IkBα and β-actin; all immunoblots are from the same samples. To detect IRF3 dimerization, WCEs were resolved by native-PAGE and analyzed by immunoblotting for IRF3. (<b>D</b>) ELISA was performed on cell culture supernatants to quantify the release of IFNβ and IFNα over time. Error bars represent SEM from two independent samples.</p