Reduced RNA Levels Research Articles

The long non-coding RNA Meg3 mediates imprinted gene expression during stem cell differentiation.

The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5'-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders.

Abstract 5364: Progesterone receptor modulates the antigen processing and presentation machinery decreasing MHC class I expression on tumors

Abstract Hormone receptor (HR) positive breast cancers, which account for approximately 75% of all breast cancers and express both estrogen receptor (ER) and progesterone receptor (PR), have been the subject of much research. As such, successful anti-estrogen/ER-based therapies have been very successful in the treatment of HR+ breast cancer. Despite this success, over 1/3 of patients will eventually progress on current ER/estrogen-targeted therapies, underscoring the need for new targeted therapies in HR+ breast cancer. Unlike ER, the role of PR in breast cancer progression has received far less attention. In a previous study, we found that a mouse mammary tumor cell line E0771, modified to express the mouse progesterone receptor (mPR) and the OVA peptide, demonstrated decreased T cell-mediated cell death when treated with progesterone compared to the control group. Progesterone-mediated protection from T-cell death was only observed in mPR+ cell lines. Flow analysis of the E0771-Ova-mPR cells also revealed a decrease in MHC Class I presentation in progesterone-treated cells compared to a vehicle control. To further explore the role of the machinery required for antigen processing and presentation of MHC Class I molecules, we conducted additional experiments using the E0771 cell line. Our results showed that treatment with progesterone led to a reduction in RNA levels of all the machinery required for antigen processing and presentation (APP). Based on these findings, we hypothesize that activation of the progesterone receptor by progesterone may decrease MHC Class I expression by regulating APP. Our results showed that treatment with progesterone resulted in a reduction in RNA levels of Tap1, Tap2, Tapbp, Nlrc5, B2m, and Psmb8, which are essential for APP. We therefore propose that activation of the progesterone receptor by progesterone may decrease MHC Class I expression by directly modulating the APP pathway. The findings of this study may have significant implications for our understanding of how breast cancer cells evade T-cell mediated cytotoxicity. Citation Format: Julio Tinoco, Lauryn Werner, Eilidh Chowanec, Harmony Saunders, Sun Xiaopeng, Jay Gertz, Justin Balko, Zachary C. Hartman, Christy Hagan. Progesterone receptor modulates the antigen processing and presentation machinery decreasing MHC class I expression on tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5364.

Immunisation efficacy of a stabilised SARS-CoV-2 spike glycoprotein in two geriatric animal models

Age is associated with reduced efficacy of vaccines and linked to higher risk of severe COVID-19. Here we determined the impact of ageing on the efficacy of a SARS-CoV-2 vaccine based on a stabilised Spike glycoprotein (S-29) that had previously shown high efficacy in young animals. Thirteen to 18-month-old golden Syrian hamsters (GSH) and 22–23-month-old K18-hCAE2 mice were immunised twice with S-29 protein in AddaVaxTM adjuvant. GSH were intranasally inoculated with SARS-CoV-2 either two weeks or four months after the booster dose, while all K18-hACE2 mice were intranasally inoculated two weeks after the second immunisation. Body weight and clinical signs were recorded daily post-inoculation. Lesions and viral load were investigated in different target tissues. Immunisation induced seroconversion and production of neutralising antibodies; however, animals were only partially protected from weight loss. We observed a significant reduction in the amount of viral RNA and a faster viral protein clearance in the tissues of immunized animals. Infectious particles showed a faster decay in vaccinated animals while tissue lesion development was not altered. In GSH, the shortest interval between immunisation and inoculation reduced RNA levels in the lungs, while the longest interval was equally effective in reducing RNA in nasal turbinates; viral nucleoprotein amount decreased in both tissues. In mice, immunisation was able to improve the survival of infected animals. Despite the high protection shown in young animals, S-29 efficacy was reduced in the geriatric population. Our research highlights the importance of testing vaccine efficacy in older animals as part of preclinical vaccine evaluation.

Nucleotide alterations in the HLA-C class I gene can cause aberrant splicing and marked changes in RNA levels in a polymorphic context-dependent manner.

Polymorphisms of HLA genes, which play a crucial role in presenting peptides with diverse sequences in their peptide-binding pockets, are also thought to affect HLA gene expression, as many studies have reported associations between HLA gene polymorphisms and their expression levels. In this study, we devised an ectopic expression assay for the HLA class I genes in the context of the entire gene, and used the assay to show that the HLA-C*03:03:01 and C*04:01:01 polymorphic differences observed in association studies indeed cause different levels of RNA expression. Subsequently, we investigated the C*03:23N null allele, which was previously noted for its reduced expression, attributed to an alternate exon 3 3' splice site generated by G/A polymorphism at position 781 within the exon 3. We conducted a thorough analysis of the splicing patterns of C*03:23N, and revealed multiple aberrant splicing, including the exon 3 alternative splicing, which overshadowed its canonical counterpart. After confirming a significant reduction in RNA levels caused by the G781A alteration in our ectopic assay, we probed the function of the G-rich sequence preceding the canonical exon 3 3' splice site. Substituting the G-rich sequence with a typical pyrimidine-rich 3' splice site sequence on C*03:23N resulted in a marked elevation in RNA levels, likely due to the enhanced preference for the canonical exon 3 3' splice site over the alternate site. However, the same substitution led to a reduction in RNA levels for C*03:03:01. These findings suggested the dual roles of the G-rich sequence in RNA expression, and furthermore, underscore the importance of studying polymorphism effects within the framework of the entire gene, extending beyond conventional mini-gene reporter assays.

Differential Outcomes of Infection by Wild-Type SARS-CoV-2 and the B.1.617.2 and B.1.1.529 Variants of Concern in K18-hACE2 Transgenic Mice.

SARS-CoV-2 is a respiratory virus with neurological complications including the loss of smell and taste, headache, and confusion that can persist for months or longer. Severe neuronal cell damage has also been reported in some cases. The objective of this study was to compare the infectivity of the wild-type virus, Delta (B.1.617.2) and Omicron (B.1.1.529) variants in transgenic mice that express the human angiotensin-converting enzyme 2 (hACE2) receptor under the control of the keratin 18 promoter (K18) and characterize the progression of infection and inflammatory response in the lungs, brain, medulla oblongata, and olfactory bulbs of these animals. We hypothesized that wild type, Delta and Omicron differentially infect K18-hACE2 mice, thereby inducing distinct cellular responses. K18-hACE2 female mice were intranasally infected with wild-type, Delta, or Omicron variants and euthanized either at 3 days post-infection (dpi) or at the humane endpoint. None of the animals infected with the Omicron variant reached the humane endpoint and were euthanized at day 8 dpi. Virological and immunological analyses were performed in the lungs, brains, medulla oblongata and olfactory bulbs isolated from infected mice. At 3 dpi, mice infected with wild type and Delta displayed significantly higher levels of viral RNA in the lungs than mice infected with Omicron, while in the brain, Delta and Omicron resulted in higher levels of viral RNA than with the wild type. Viral RNA was also detected in the medulla oblongata of mice infected by all these virus strains. At this time point, the mice infected with wild type and Delta displayed a marked upregulation of many inflammatory markers in the lungs. On the other hand, the upregulation of inflammatory markers was observed only in the brains of mice infected with Delta and Omicron. At the humane endpoint, we observed a significant increase in the levels of viral RNA in the lungs and brains of mice infected with wild type and Delta, which was accompanied by the elevated expression of many inflammatory markers. In contrast, mice which survived infection with the Omicron variant showed high levels of viral RNA and the upregulation of cytokine and chemokine expression only in the lungs at 8 dpi, suggesting that infection and inflammatory response by this variant is attenuated in the brain. Reduced RNA levels and the downregulation of inflammatory markers was also observed in the medulla oblongata and olfactory bulbs of mice infected with Omicron at 8 dpi as compared with mice infected with wild-type and Delta at the humane end point. Collectively, these data demonstrate that wild-type, Delta, and Omicron SARS-CoV-2 induce distinct levels of infection and inflammatory responses in K18-hACE2 mice. Notably, sustained brain infection accompanied by the upregulation of inflammatory markers is a critical outcome in mice infected with wild type and Delta but not Omicron.

Carnitine palmitoyl-transferase 1A is potentially involved in bovine herpesvirus 1 productive infection

Bovine herpesvirus 1(BoHV-1) is an important bovine pathogen that causes great economic loss to cattle farms worldwide. The virus-productive infection in bovine kidney (MDBK) cells results in ATP depletion. The mechanisms are not well understood. Mitochondrial fatty acid β-oxidation (FAO) is an important energy source in many tissues with high energy demand. Since carnitine palmitoyl-transferase 1 A (CPT1A) is the rate-limiting enzyme of FAO, we investigated the interactions between virus-productive infection and CPT1A signaling. Here, we found that virus-productive infection at the later stage significantly decreased CPT1A protein levels in all the detected cells, including MDBK, A549, and Neuro-2A cells, differentially altered the accumulation of CPT1A proteins in the nucleus and cytosol, and re-localized the protein in the nucleus. Etomoxir (ETO), an irreversible inhibitor of CPT1A, inhibited viral replication and partially interfered with the ability of BoHV-1 to alter CPT1A accumulation in the nucleus but not in the cytosol. Furthermore, ETO consistently reduced RNA levels of two viral regulatory proteins (bICP0 and bICP22) and protein expression of virion-associated proteins during productive infection, further supporting the important roles of CPT1A signaling in BoHV-1 productive infection. These data, for the first time, suggest that CPT1A is potentially involved in BoHV-1 productive infection.

Loratadine inhibits Staphylococcus aureus virulence and biofilm formation

SummaryThere are no anti-virulence and anti-biofilm treatments for Staphylococcus aureus infection. We found that 25 μM loratadine inhibits S. aureus biofilm formation under static or flow-based conditions. Testing of loratadine effects on 255 clinical S. aureus strains with varying biofilm robustness showed inhibition of biofilm formation in medium and strong, but not weak, biofilm-producing strains. At 25 μM, loratadine reduced pigmentation and hemolysis of the bacteria without affecting growth. Loratadine (5 mg/kg) reduced mortality in S. aureus pulmonary infection model mice and acted synergistically with vancomycin to reduce pulmonary bacterial load and levels of inflammatory cytokines in bronchoalveolar lavage fluid. Loratadine analogues (side-chain carbamate moiety changed) inhibited biofilm formation, pigmentation, and hemolysis of S. aureus. Regarding mechanism, loratadine exposure reduced RNA levels of virulence-related S. aureus genes, and loratadine-induced mutations in MgrA reduced loratadine-MgrA binding. Overexpression of mutated mgrA in wild-type S. aureus decreased the biofilm formation inhibition effect of loratadine.

CRISPR guides induce gene silencing in plants in the absence of Cas

BackgroundRNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation, and expanded function including antiviral immunity.ResultsHere, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we observe that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. Small RNA sequencing during GIGS identifies the production of small RNA that extend beyond the crRNA expressed sequence in samples expressing multi-guide crRNA. Additionally, we demonstrate that mismatches in guide sequences at position 10 and 11 abolish GIGS. Finally, we show that GIGS is elicited by guides that lack the Cas13 direct repeat and can extend to Cas9 designed crRNA of at least 28 base pairs, indicating that GIGS can be elicited through a variety of guide designs and is not dependent on Cas13 crRNA sequences or design.ConclusionsCollectively, our results suggest that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins, or long double-stranded RNA. Given similar evidence of Cas13-independent silencing in an insect system, it is likely GIGS is active across many eukaryotes. Our results show that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement and functional genomics.

Environmentally contingent control of Candida albicans cell wall integrity by transcriptional regulator Cup9.

The fungal pathogen Candida albicans is surrounded by a cell wall that is the target of caspofungin and other echinocandin antifungals. Candida albicans can grow in several morphological forms, notably budding yeast and hyphae. Yeast and hyphal forms differ in cell wall composition, leading us to hypothesize that there may be distinct genes required for yeast and hyphal responses to caspofungin. Mutants in 27 genes reported previously to be caspofungin hypersensitive under yeast growth conditions were all caspofungin hypersensitive under hyphal growth conditions as well. However, a screen of mutants defective in transcription factor genes revealed that Cup9 is required for normal caspofungin tolerance under hyphal and not yeast growth conditions. In a hyphal-defective efg1Δ/Δ background, Cup9 is still required for normal caspofungin tolerance. This result argues that Cup9 function is related to growth conditions rather than cell morphology. RNA-seq conducted under hyphal growth conditions indicated that 361 genes were up-regulated and 145 genes were down-regulated in response to caspofungin treatment. Both classes of caspofungin-responsive genes were enriched for cell wall-related proteins, as expected for a response to disruption of cell wall integrity and biosynthesis. The cup9Δ/Δ mutant, treated with caspofungin, had reduced RNA levels of 40 caspofungin up-regulated genes, and had increased RNA levels of 8 caspofungin down-regulated genes, an indication that Cup9 has a narrow rather than global role in the cell wall integrity response. Five Cup9-activated surface-protein genes have roles in cell wall integrity, based on mutant analysis published previously (PGA31 and IFF11) or shown here (ORF19.3499, ORF19.851, or PGA28), and therefore may explain the hypersensitivity of the cup9Δ/Δmutant to caspofungin. Our findings define Cup9 as a new determinant of caspofungin susceptibility.

Genomic and Transcriptomic Investigation of the Physiological Response of the Methylotroph Bacillus methanolicus to 5-Aminovalerate.

The methylotrophic thermophile Bacillus methanolicus can utilize the non-food substrate methanol as its sole carbon and energy source. Metabolism of L-lysine, in particular its biosynthesis, has been studied to some detail, and methanol-based L-lysine production has been achieved. However, little is known about L-lysine degradation, which may proceed via 5-aminovalerate (5AVA), a non-proteinogenic ω-amino acid with applications in bioplastics. The physiological role of 5AVA and related compounds in the native methylotroph was unknown. Here, we showed that B. methanolicus exhibits low tolerance to 5AVA, but not to related short-chain (C4–C6) amino acids, diamines, and dicarboxylic acids. In order to gain insight into the physiological response of B. methanolicus to 5AVA, transcriptomic analyses by differential RNA-Seq in the presence and absence of 5AVA were performed. Besides genes of the general stress response, RNA levels of genes of histidine biosynthesis, and iron acquisition were increased in the presence of 5AVA, while an Rrf2 family transcriptional regulator gene showed reduced RNA levels. In order to test if mutations can overcome growth inhibition by 5AVA, adaptive laboratory evolution (ALE) was performed and two mutants—AVA6 and AVA10—with higher tolerance to 5AVA were selected. Genome sequencing revealed mutations in genes related to iron homeostasis, including the gene for an iron siderophore-binding protein. Overexpression of this mutant gene in the wild-type (WT) strain MGA3 improved 5AVA tolerance significantly at high Fe2+ supplementation. The combined ALE, omics, and genetics approach helped elucidate the physiological response of thermophilic B. methanolicus to 5AVA and will guide future strain development for 5AVA production from methanol.

Loratadine Interacts With MgrA and Inhibits <i>Staphylococcus aureus</i> Virulence and Biofilm Formation

There are no anti-virulence and anti-biofilm treatments for Staphylococcus aureus infection. We found that 25 μM loratadine inhibits S. aureus biofilm formation under static or flow-based conditions. Testing of loratadine effects on 255 clinical S. aureus strains with varying biofilm robustness, showed inhibition of biofilm formation in medium and strong, but not weak, biofilm-producing strains. At 25 μM, loratadine reduced pigmentation and hemolysis of the bacteria without affecting growth. Loratadine (5 mg/kg) reduced mortality in S. aureus pulmonary-infection model mice, and acted synergistically with vancomycin to reduce pulmonary bacterial load and levels of inflammatory cytokines in bronchoalveolar lavage fluid. Loratadine analogues (side-chain carbamate moiety changed) inhibited biofilm formation, pigmentation, and hemolysis of S. aureus. Regarding mechanism, loratadine exposure reduced RNA levels of virulence-related S. aureus genes, and loratadine-induced mutations in MgrA reduced loratadine-MgrA binding. Loratadine medical-implant coating may be an effective prophylaxis against S. aureus virulence and biofilm establishment.

Abstract PO-134: Intracellular calcium regulates androgen receptor expression and basal-luminal features in prostate cancer from African American men

Abstract Background: Disparities in prostate cancer (PrCa) incidence and mortality between African American (AA) and European American (EA) men are partially mediated by underlying disease biology. The goal of this study was to determine how DNA methylation and transcriptomic changes drive PrCa health disparities in AA men. Methods: Transcriptomic alterations potentially mediated by DNA methylation were identified by Illumina arrays and RNA-sequencing in PrCa from 30 AA and 32 EA men. Androgen receptor (AR) protein expression was measured using tissues microarray of matched PrCa tissues from 95 EA and 92 AA men. In vitro, MDA PCa 2a (2a)/MDA PCa 2b (2b) cells derived from an AA man and LNCaP/LaPC-4 cells derived from EA men were stimulated with ionomycin, a calcium ionophore to measure intracellular calcium using fluorescent-based live imaging. Results: Unsupervised hierarchical clustering revealed DNA methylation clusters (Cluster A and Cluster B) with differential methylation of loci that regulate intracellular calcium levels including RYR2, TRPC6, and TRPA1. Increased methylation of calcium regulatory genes in Cluster A was associated with reduced disease-free time (DFT) (21.65 vs 46.71 months, p<0.05) only in AA men with PrCa. RYR2 (-0.122 vs -0.004, p=0.69), TRPC6 (0.006 vs -0.639, p=0.06) and TRPA1 (-0.070 vs -0.269, p<0.05) transcript levels were lower in Cluster A compared to Cluster B. These data suggest DNA methylation can regulate expression of calcium regulating genes. In vitro, we found AA PrCa derived 2b cells have reduced RNA levels of RYR2, TRPV6 and CALB1 compared to EA PrCa derived LNCaP cells. Reduced transcription can result in lower protein expression and thus activity of calcium regulatory genes. To test this, we stimulated cells with ionomycin and found a rapid increase in intracellular calcium in 2a/2b cells (within 60 seconds) compared to LNCaP/LAPC-4 cells (120-300 seconds). This suggests that decreased transcription correlates to reduced buffering capacity of calcium regulatory genes. Others have shown that increased intracellular calcium reduces AR protein levels. Therefore, we analyzed AR protein expression in a subset of tumors from Cluster A and B. AR levels were lower in adjacent non-tumor and tumor tissue in these overlapping samples. AR low PrCa are associated with basal like features and respond poorly to androgen deprivation therapy. Therefore, we analyzed the PAM50 basal/luminal gene sets and AR target genes and found differentially expression of these genes in AA PrCa derived 2b and EA PrCa derived LNCaP cells. Conclusions: Our study shows that AA men with PrCa have epigenetically dysregulated calcium signaling that is associated with worse DFT. Our ongoing work seeks to identify how these alterations regulate AR expression and thus basal/luminal features. Our long- term goal is to establish novel molecular subtypes using calcium-AR-basal/luminal features that can guide design of rationale therapies in a subset of AA men with PrCa. Citation Format: Swathi Ramakrishnan, Xuan Peng, Eduardo C. Gomez, Kristopher Attwood, Ivan V. Maly, Wilma A. Hoffmann, Wendy Huss, Gissou Azabdaftari, Li Yan, Jianmin Wang, Anna Woloszynska. Intracellular calcium regulates androgen receptor expression and basal-luminal features in prostate cancer from African American men [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-134.

SUN-011 Identification Of Regulators Of Estrogenic Signalling In ER-positive Breast Cancer Cells By Whole Genome shRNA Screening

Over 70% of breast tumours express estrogen receptor alpha (ERα), a ligand-inducible transcription factor. Stimulation with estradiol leads to receptor binding to estrogen response elements (EREs) in target genes regulatory sequences in association with transcriptional cofactors. This results in altered gene expression, increased cell proliferation and accelerated tumour growth. To systematically identify genes contributing to ERα expression and signalling in breast cancer, we performed a genome-wide screen in human ERα+ luminal breast cancer cell lines stably expressing estrogen-induced luciferase reporters using an arrayed format. A primary screen (Sigma Mission lentiviral library, 16083 genes, three shRNA/gene) and secondary screens (615 genes identified as hits by at least two shRNAs with concordant impact on reporter gene expression without major effects on cell survival) were carried out using estrogen-sensitive cells (T47D-ERE3-Luc, MCF-7-ERE-Luc) and insensitive controls (antioxidant response reporter T47D-ARE-Luc). We ultimately characterized by RNASeq the impact of suppressing expression of each of 30 prioritized hits (2-3 shRNAs with >50% reduction of RNA levels per hit) on gene expression in ERα+ breast cancer cells. Knockdown of several of these hits had extensive impacts on expression of direct estrogen target genes and on ERα+ breast cancer cell proliferation. Some hits regulated ERα gene expression and several interacted with ERα in co-IP experiments, indicating roles as upstream regulators and/or transcriptional cofactors of ERα.

Disruption of peroxisome proliferator–activated receptor γ coactivator (PGC)-1α reverts key features of the neoplastic phenotype of glioma cells

The peroxisome proliferator-activated receptor γ coactivator (PGC)-1α is a master regulator of mitochondrial biogenesis and controls metabolism by coordinating transcriptional events. Here, we interrogated whether PGC-1α is involved in tumor growth and the metabolic flexibility of glioblastoma cells. PGC-1α was expressed in a subset of established glioma cell lines and primary glioblastoma cell cultures. Furthermore, a higher PGC-1α expression was associated with an adverse outcome in the TCGA glioblastoma dataset. Suppression of PGC-1α expression by shRNA in the PGC-1α-positive U343MG glioblastoma line suppressed mitochondrial gene expression, reduced mitochondrial membrane potential, and diminished oxygen as well as glucose consumption, and lactate production. Compatible with the known PGC-1α functions in reactive oxygen species (ROS) metabolism, glioblastoma cells deficient in PGC-1α displayed ROS accumulation, had reduced RNA levels of proteins involved in ROS detoxification, and were more susceptible to death induction by H2O2 compared with control cells. PGC-1αsh cells also had impaired proliferation and migration rates in vitro and displayed less stem cell characteristics. Complementary effects were observed in PGC-1α-low LNT-229 cells engineered to overexpress PGC-1α. In an in vivo xenograft experiment, tumors formed by U343MG PGC-1αsh glioblastoma cells grew much slower than control tumors and were less invasive. Interestingly, the PGC-1α knockdown conferred protection against hypoxia-induced cell death, probably as a result of less active anabolic pathways, and this effect was associated with reduced epidermal growth factor expression and mammalian target of rapamycin signaling. In summary, PGC-1α modifies the neoplastic phenotype of glioblastoma cells toward more aggressive behavior and therefore makes PGC-1α a potential target for anti-glioblastoma therapies.

PTEN/PI3k/AKT Regulates Macrophage Polarization in Emphysematous mice.

Macrophages play an important role in the pathogenesis of COPD. Macrophage polarization towards the M2 phenotype has been observed in the lung tissues of COPD patients and cigarette smokers. The molecular basis of this process remains unclear, and it has not been completely illuminated in animal models of emphysema. In our study, we combined cigarette smoke (CS) exposure with intraperitoneal injection of cigarette smoke extract (CSE) to build an emphysema model. We found by immunohistochemical staining and flow cytometry that the expression level of CD206 and the ratio of M2 to M1 macrophages was increased in emphysematous mice. We also demonstrated that decreased protein level for phosphatase and tensin homology deleted on chromosome ten (PTEN) and increased total protein levels for phosphorylation -protein kinase B (p-AKT) in the lung tissue of emphysematous mice and in CSE-treated RAW264.7 cells. In both bone marrow-derived macrophages (BMDMs) from emphysematous mice and CSE-treated RAW264.7 cells, we observed by RT-PCR that the mRNA levels of M2 macrophage-related markers and cytokines were increased. Furthermore, M1 macrophage-related markers and cytokines were decreased. Meanwhile we treated BMDMs from emphysematous mice and CSE-treated RAW264.7 cells with the phosphoinositide 3-kinase (PI3K)/Akt inhibitor (LY294002), we observed a reduction in RNA levels of M2 macrophage-related markers and cytokines. In conclusion, we confirmed that macrophage M2 polarization was induced in emphysematous mice generated by CS exposure combined with intraperitoneal injection of CSE. We also showed that M2 polarization was mediated through PTEN/PI3k/AKT pathway activation.

P01.11 The transcriptional coactivator PGC-1a promotes defence against reactive oxygen species (ROS) and proliferation in human glioma cells

AbstractGene expression of many signalling pathways and metabolic programs is regulated at the transcriptional level by DNA binding coactivators. These coactivators act in a tissue-specific-manner and are responsive to different physiological stimuli like nutrient supply and temperature. One of these coactivators, Peroxisome proliferator-activated receptor- coactivator (PGC)-1α, is a master regulator of mitochondrial biogenesis, controls antioxidative responses and coordinates posttranscriptional events. The activity of PGC-1a is regulated by growth factor receptor signaling and the AMP-activated protein kinase (AMPK) pathway, both of which are crucial for the growth and metabolism of tumors. Further, because variable nutritional environments require tumor cells to adapt their energy metabolism, we asked whether PGC-1α is involved in growth, resistance against nutrient depletion and metabolic flexibility of glioma cells.We found that PGC-1α is expressed in a subset of established and primary glioma cells. Surprisingly, while suppression of PGC-1α expression by sh-RNA in U343MG glioma cells reduced glucose consumption and lactate production, it did not reduce oxygen consumption or mitochondrial mass. PGC-1α-sh cells had impaired proliferation and migration in vitro. Compatible to the known PGC-1α’s functions in reactive oxygen species (ROS) metabolism, glioma cells lacking PGC-1α expression had reduced RNA levels of proteins involved in (ROS) detoxification mechanism, and these cells were more susceptible to death induction by H2O2 compared to control cells. Interestingly, the PGC-1α-knock-down conferred protection against hypoxia-induced cell death. In an in-vivo-xenograft experiment, tumors formed by PGC-1α-suppressed glioma cells grew much slower than control tumors.In summary, PGC-1α is expressed in glioma cells and has an important role in growth and resistance against ROS in these cells. As the oxygen consumption in PGC-1α-sh cells was not significantly impaired, the presented effects of PGC-1α seemingly do not depend on the well-established functions of PGC-1α on mitochondrial oxidative phosphorylation. Further experiments are underway to analyse in more detail the metabolic and cellular functions of PGC-1α.

Abstract POSTER-THER-1428: Effects of atypical PKC inhibitors on ovarian cancer proliferation and RNA levels

Abstract Ovarian cancers are highly lethal tumors which account for approximately four percent of all women’s cancers and are the fifth leading cause of cancer-related death among women. At diagnosis the majority of patients have metastatic disease and the long-term survival remains low. Certain ovarian cancers are highly lethal tumors due to the emergence of therapy-resistant ovarian cancer cells. Protein kinase C-iota (PKC-ι) has been shown to aid in the ability of cancer cells to resist drug-induced apoptosis. Recently, it has been reported that PKC-ι which is located in chromosome 3 at 3q26.2 is the most common genomic amplicon as identified by comparative genomic hybridization [Proc Natl Acad Sci U S A 102:12519–12524(2005)]. Additionally, an increase in PKC-ι DNA copy number was associated with decreased progression-free survival of ovarian cancer patients. Moreover, only PKC-ι gene amplification is highly correlated with protein overexpression, tumor size, lymph node metastasis and clinical stage out of four genes studied on the 3q26 amplification [Genes Chromosomes Cancer 47:127-136(2008)]. The focus of this research was to determine the in-vitro efficacy of the pan-atypical PKC inhibitor, 2-acetyl-1,3-cyclopentanedione (ACPD; Diabetes. 2014 Apr 4) and the novel PKC-iota inhibitor, ICA-1, (1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy) methyl]cyclopentyl]-,[1R-(1α, 2β, 3 β, 4 α) [The Inter. J. Biochem. & Cell Biol. 43:784-794(2011)] on HEY ovarian cancer cell proliferation and RNA concentration. In contrast to ACPD which inhibits both PKC-ι and PKC-zeta (PKC-ζ), ICA-1 specifically inhibits the activity of PKC-ι but not (PKC-ζ). The effects of ACPD and ICA-1on HEY proliferation was quantified by Trypan blue exclusion assay. RNA was isolated from treated and untreated cells using the Rneasy Mini Kit and protocol. Total RNA concentration was estimated using the NanoDrop1000 Spectrophotometer. Results showed that incubation of HEY ovarian cancer cells with ICA-1(1 μm) decreased proliferation by 41% compared to controls at 72 hours post-treatment. ICA-1(2 μM) reduced RNA levels by 17% at 72 hours post-treatment compared to controls. In contrast, ACPD (2 μM) inhibited proliferation by 82% compared to controls and RNA levels were reduced by 97% at 24 hours post-treatment. These results suggest the potential of ICA-1 and ACPD as chemotherapeutic agents. Citation Format: Minjel Shah, Christopher Apostolatos, Hercules Apostolatos, Mildred Acevedo-Duncan. Effects of atypical PKC inhibitors on ovarian cancer proliferation and RNA levels [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1428.

Poly(A)-specific ribonuclease deficiency impacts telomere biology and causes dyskeratosis congenita.

Dyskeratosis congenita (DC) and related syndromes are inherited, life-threatening bone marrow (BM) failure disorders, and approximately 40% of cases are currently uncharacterized at the genetic level. Here, using whole exome sequencing (WES), we have identified biallelic mutations in the gene encoding poly(A)-specific ribonuclease (PARN) in 3 families with individuals exhibiting severe DC. PARN is an extensively characterized exonuclease with deadenylation activity that controls mRNA stability in part and therefore regulates expression of a large number of genes. The DC-associated mutations identified affect key domains within the protein, and evaluation of patient cells revealed reduced deadenylation activity. This deadenylation deficiency caused an early DNA damage response in terms of nuclear p53 regulation, cell-cycle arrest, and reduced cell viability upon UV treatment. Individuals with biallelic PARN mutations and PARN-depleted cells exhibited reduced RNA levels for several key genes that are associated with telomere biology, specifically TERC, DKC1, RTEL1, and TERF1. Moreover, PARN-deficient cells also possessed critically short telomeres. Collectively, these results identify a role for PARN in telomere maintenance and demonstrate that it is a disease-causing gene in a subset of patients with severe DC.

Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.

Early presentation of cystic kidneys in a family with a homozygous INVS mutation

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease that is the most frequent monogenic cause of end-stage renal disease in children. Infantile NPHP, often in combination with other features like situs inversus, are commonly caused by mutations in the INVS gene. INVS encodes the ciliary protein inversin, and mutations induce dysfunction of the primary cilia. In this article, we present a family with two severely affected fetuses that were aborted after discovery of grossly enlarged cystic kidneys by ultrasonography before 22 weeks gestation. Exome sequencing showed that the fetuses were homozygous for a previously unreported nonsense mutation, resulting in a truncation in the IQ1 domain of inversin. This mutation induces nonsense-mediated RNA decay, as suggested by a reduced RNA level in fibroblasts derived from the fetus. However, a significant amount of mutant INVS RNA was present in these fibroblasts, yielding mutant inversin protein that was mislocalized. In control fibroblasts, inversin was present in the ciliary axoneme as well as at the basal body, whereas in the fibroblasts from the fetus, inversin could only be detected at the basal body. The phenotype of both fetuses is partly characteristic of infantile NPHP and Potter sequence. We also identified that the fetuses had mild skeletal abnormalities, including shortening and bowing of long bones, which may expand the phenotypic spectrum associated with INVS mutations.