Post-transcriptional Down-regulation Research Articles

Posttranscriptional Upregulation by MicroRNAs

MicroRNAs are small non-coding RNA guide molecules that regulate gene expression via association with effector complexes and sequence-specific recognition of target sites on other RNAs; misregulated microRNA expression and functions are linked to a variety of tumors, developmental disorders, and immune disease. MicroRNAs have primarily been demonstrated to mediate posttranscriptional downregulation of expression; translational repression, and deadenylation-dependent decay of messages through partially complementary microRNA target sites in mRNA untranslated regions (UTRs). However, an emerging assortment of studies, discussed in this review, reveal that microRNAs and their associated protein complexes (microribonucleoproteins or microRNPs) can additionally function to posttranscriptionally stimulate gene expression by direct and indirect mechanisms. These reports indicate that microRNA-mediated effects can be selective, regulated by the RNA sequence context, and associated with RNP factors and cellular conditions. Like repression, translation upregulation by microRNAs has been observed to range from fine-tuning effects to significant alterations in expression. These studies uncover remarkable, new abilities of microRNAs and associated microRNPs in gene expression control and underscore the importance of regulation, in cis and trans, in directing appropriate microRNP responses.

A COMT gene haplotype associated with methamphetamine abuse

Methamphetamine (MAMP) use is highly associated with psychiatric disorders with 12-13% of MAMP-dependent patients experiencing psychotic symptoms. Substance abuse and dependence may primarily involve the mesolimbic pathway and dopaminergic brain structures. It follows that dopaminergic genes, particularly COMT (encoding catechol-O-methyltransferase) and its val158met polymorphism (rs4680), are natural candidates for susceptibility loci to addiction. We have previously found an association with rs4680 and MAMP addiction. We present additional genotyping of rs165599 in 423 cases and 502 controls of a Taiwanese MAMP user sample. We carried out an in-silico evaluation of rs165599 for a possible impact on microRNA binding or UTR stability. We also carried out a review of transcript sequences across the COMT 3'UTR. Genotype counts were (cases/controls): AA 94/110, AG 198/210 and GG 93/109. There were no significant allele or genotype differences between cases and controls for rs165599. However, a haplotype main effect was detected using both rs4680 and rs165599 using the χ²-test in UNPHASED. The global P-value was P=0.0044 with the effect appearing to derive from one haplotype that is underrepresented in cases: A/G for rs4680/rs165599 (haplotype P=0.001). rs165599 is a single nucleotide polymorphism located in the COMT 3' untranslated region (UTR), a noncoding transcript region subject to posttranscriptional down-regulation by mechanisms such as microRNA binding. A review of transcript sequences across the COMT 3'UTR found evidence to suggest antisense interference of COMT from the 3'UTR of the neighbouring 'Armadillo repeat gene deleted in velocardiofacial syndrome' gene.

The Antiepileptic Drugs Phenobarbital and Carbamazepine Reduce Transport of Methotrexate in Rat Choroid Plexus by Down-Regulation of the Reduced Folate Carrier

Intrathecal methotrexate (MTX) has been associated with severe neurotoxicity. Because carrier-associated removal of MTX from the cerebrospinal fluid (CSF) into blood remains undefined, we determined the expression and function of MTX transporters in rat choroid plexus (CP). MTX neurotoxicity usually manifests as seizures requiring therapy with antiepileptic drugs (AEDs) such as phenobarbital (PB). Because we have demonstrated that PB reduces activity of MTX influx carrier reduced folate carrier (Rfc1) in liver, we investigated the influence of the AEDs PB, carbamazepine (CBZ), or gabapentin on Rfc1-mediated MTX transport in CP. Reverse transcriptase-polymerase chain reaction and Western blot analysis showed similar expression of the MTX influx carrier Rfc1 and organic anion transporter 3 or efflux transporter multidrug resistance-associated protein 1 (Mrp1) and breast cancer resistance protein (Bcrp) in rat CP tissue and choroidal epithelial Z310 cells. Confocal microscopy revealed subcellular localization of Rfc1 and Bcrp at the apical and of Mrp1 at the basolateral CP membrane. Uptake, efflux, and inhibition studies indicated MTX transport activity of Rfc1, Mrp1, and Bcrp. PB and CBZ but not gabapentin significantly inhibited Rfc1-mediated uptake of MTX in CP cells. Studies on the regulatory mechanism showed that PB significantly inhibited Rfc1 translation but did not alter carrier gene expression. Altogether, removal of intrathecal MTX across the blood-CSF barrier may be achieved through Rfc1-mediated uptake from the CSF followed by MTX extrusion into blood, particularly via Mrp1. Antiepileptic treatment with PB or CBZ causes post-transcriptional down-regulation of Rfc1 activity in CP. This mechanism may result in enhanced MTX toxicity in patients with cancer who are receiving intrathecal MTX chemotherapy by reduced CSF clearance of the drug.

Akt Stimulates Hepatic SREBP1c and Lipogenesis through Parallel mTORC1-Dependent and Independent Pathways

Through unknown mechanisms, insulin activates the sterol regulatory element-binding protein (SREBP1c) transcription factor to promote hepatic lipogenesis. We find that this induction is dependent on the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). To further define the role of mTORC1 in the regulation of SREBP1c in the liver, we generated mice with liver-specific deletion of TSC1 (LTsc1KO), which results in insulin-independent activation of mTORC1. Surprisingly, the LTsc1KO mice are protected from age- and diet-induced hepatic steatosis and display hepatocyte-intrinsic defects in SREBP1c activation and de novo lipogenesis. These phenotypes result from attenuation of Akt signaling driven by mTORC1-dependent insulin resistance. Therefore, mTORC1 activation is not sufficient to stimulate hepatic SREBP1c in the absence of Akt signaling, revealing the existence of an additional downstream pathway also required for this induction. We provide evidence that this mTORC1-independent pathway involves Akt-mediated suppression of Insig2a, a liver-specific transcript encoding the SREBP1c inhibitor INSIG2.

Abstract 270: The TWIST1-microRNA-22 axis represses estrogen receptor alpha expression in breast cancer

Abstract Background: TWIST1 (Twist) is a master regulator of embryonic cell fate specification and differentiation. It mediates its transrepressive and transactivating functions by binding to consensus E-box sequences within promoters. Importantly, ectopic expression of Twist in breast epithelial tissue is an initiator of breast cancer. Estrogen and estrogen receptor (ER) expression play a crucial role in breast cancer. ER expression is regulated by methylation, chromatin inactivation, alternative promoter utilization, mutations, as well as cis and trans-acting elements. MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression. We show here that Twist binds to E-boxes in the promoter of the microRNA miR-22 and upregulates its expression in breast cancer. Furthermore, the dysregulated miR-22 binds to the 3’-UTR of ER and downregulates its expression leading to an ER negative state. Methods: We isolated miRNA from breast cancer cells and assayed for miR-22 expression by qRT-PCR. A miR-22 construct was transfected into breast cancer cells to force expression of miR-22 and analyze effects on ER. Inhibition studies were carried out using targeted knockdowns of miR-22 and analyzed by qRT-PCR. Fine mapping of the miR-22 genomic structure was performed by RACE. The miR-22 promoter containing three E-boxes was cloned and used in dual luciferase assays to study Twist binding and miR-22 activation. Chromatin immunoprecipitation assays were carried out to confirm Twist binding. The 3’-UTR of the ER was cloned into the pMIR-Report vector and the construct used to study miR-22 binding. Functionality of miR-22 binding to the ER 3’-UTR leading to loss of ER expression was confirmed by immunoblotting. Results: We found that miR-22 was highly expressed in cell lines expressing Twist and that ER expression was inversely proportional to miR-22 in all cancer cell lines. We discovered that miR-22 gene consists of three exons spanning over 4.5 kb in the C17orf91 putative gene in chromosome 17. Forced expression of Twist increased miR-22 expression while knockdown of Twist lowered miR-22 expression in vitro. Consequently, forced expression of Twist and miR-22 lowered expression of ER in MCF-7 as seen at both RNA and protein levels. Mechanistically, both miR-22 binding sites in the ER 3’-UTR were important for miR-22 functionality and removal of either site was sufficient to abrogate ER downregulation. Finally, we observed that miR-22 expression was inversely proportional to ER expression and directly proportional to Twist expression in human breast cancer samples. Conclusion: These results suggest that Twist repression of ER is mediated by miR-22. The mechanism is by direct binding of Twist to E-boxes in the miR-22 promoter causing it to be upregulated. The dysregulated miR-22 binds to the 3’-UTR of ER leading to post-transcriptional downregulation and loss of ER. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 270. doi:10.1158/1538-7445.AM2011-270

Abstract 3996: MicroRNA-96 is a posttranscriptional suppressor of anaplastic lymphoma kinase expression in human cancer

Abstract Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is normally expressed in human neural cells at an early developmental stage. In contrast, the expression of ALK is minimal in adult human tissues. Nonetheless, ALK is aberrantly expressed in some of the most aggressive types of adults and children malignancies. For instance, the expression of ALK in the form of the chimeric proteins NPM-ALK and EML4-ALK is seen in a subset of T-cell lymphoma and non-small lung cancer, respectively. In addition, aberrant upregulation of full-length ALK occurs in a group of neuroblastoma patients. ALK has been shown to contribute significantly to the survival of cancer cells. It is not surprising, therefore, that targeting ALK is now considered a promising therapeutic strategy to be utilized for the treatment of these malignant diseases in the near future. Although the aberrant expression of ALK has been partially attributed to the occurrence of chromosomal abnormalities that induce the expression of ALK chimeric proteins, the mechanisms underlying this interesting phenomenon are not completely identified. We reasoned that one potential mechanism contributing to the aberrant expression of ALK in cancer cells is mediated through defects in the microRNA (miR) system. We performed analysis of miR predicted targets using 3 web-based algorithms including TargetScan, miRanda, and PicTars. Mir-96 was the only candidate with potential ability to bind to ALK 3′-untranslated region (3′-UTR) that was identified simultaneously by the 3 algorithms. qPCR analysis showed that the expression of miR-96 was markedly decreased in NPM-ALK-expressing T-cell lymphoma cell lines compared with normal human T lymphocytes. The expression of miR-96 was also negligible in lung cancer and neuroblastoma cell lines that express EML4-ALK and full-length ALK, respectively. Importantly, transfection of ALK-expressing cancer cell lines with miR-96 was associated with a pronounced decrease in the expression of the different forms of ALK protein, and not in ALK mRNA. There was a marked reduction in luciferase activity when miR-96 was transfected with ALK 3′-UTR containing the presumed binding site for miR-96. Transfection of ALK-expressing cell lines with miR-96 was associated with decreased phosphorylation of ALK downstream targets, including pSTAT3, pAKT, and pIGF-IR, with no effect on the basal levels of these oncogenic proteins. Furthermore, miR-96 decreased the proliferation of cancer cell lines that express ALK. The effects of miR-96 were not detected when cell lines were transfected with a control miR mimic. In conclusion, our results identify miR-96 as a novel tumor suppressor that functions by inducing posttranscriptional downregulation of ALK expression, and suggest that miR-96 could represent a potential modality to successfully treat aggressive tumors that express the different forms of ALK protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3996. doi:10.1158/1538-7445.AM2011-3996

The conformation of microRNA seed regions in native microRNPs is prearranged for presentation to mRNA targets

MicroRNAs control gene expression by post-transcriptional down-regulation of their target mRNAs. Complementarity between the seed region (nucleotides 2–8) of a microRNA and the 3′-UTR of its target mRNA is the key determinant in recognition. However, the structural basis of the ability of the seed region to dominate target recognition in eukaryotic argonaute complexes has not been directly demonstrated. To better understand this problem, we performed chemical probing of microRNAs held in native argonaute-containing complexes isolated from Caenorhabditis elegans. Direct probing of the RNA backbone in isolated native microRNP complexes shows that the conformation of the seed region is uniquely constrained, while the rest of the microRNA structure is conformationally flexible. Probing the Watson–Crick edges of the bases shows that bases 2–4 are largely inaccessible to solvent, while seed region bases 5–8 are readily modified; collectively our probing results suggest a model in which these bases are primed for initiating base pairing with the target mRNA. In addition, an unusual DMS reactivity with U at position 6 is observed. We propose that interaction of miRNAs with argonaute proteins pre-organizes the structure of the seed sequence for specific recognition of target mRNAs.

Cannabinoid CB1 Receptor Is Downregulated in Clear Cell Renal Cell Carcinoma

Several studies in cell cultures and in animal models have demonstrated that cannabinoids have important antitumoral properties. Because many of these effects are mediated through cannabinoid (CB) receptors CB₁ and CB₂, the study of their expression in human neoplasms has become of great interest in recent years. Fresh and formalin-fixed tissue samples of 20 consecutive clear cell renal cell carcinomas (CCRCCs) were collected prospectively and analyzed for the expression of both CB receptors by using RT-PCR, Western blot (WB), and immunohistochemical techniques. RT-PCR assays demonstrated the expression of mRNA encoding the CB₁ in tumor tissue and in adjacent non-neoplastic kidney. Conversely, WB and IHC revealed a marked downregulation of CB₁ protein in tumor tissue; CB₂ was not expressed. The obtained data suggest a possible implication of the endocannabinoid system in renal carcinogenesis. A posttranscriptional downregulation of CB₁ and the absence of expression of CB₂ characterize CCRCC.

Novel Protein Encoded by C6orf105 Regulates Tissue Factor Pathway Inhibitor Expression and Function In Human Endothelial Cells In Normal Conditions and During Androgen Stimulation

AbstractAbstract 348Cardiovascular disease (CVD) and thrombotic complications (deep vein thrombosis/venous thromboembolism, DVT/VTE) represent major health problems, with men having higher rates of clinical events than women. Tissue Factor Pathway Inhibitor (TFPI) is the key natural inhibitor of coagulation: it neutralizes factor Xa (FXa) and inhibits tissue factor-factor VIIa (TF-FVIIa) in the presence of FXa. In vivo most of TFPI is in endothelial cells (EC), reversibly bound to yet unidentified receptors, and glycosyl phosphatidylinositol-floated in caveolae and/or lipid raft microdomains. Intravascular thrombosis occurs frequently in older people, especially associated with cancer, diabetes, or CVD. TF is directly involved in tumor hypercoagulability, angiogenesis and metastasis. Cell-associated TFPI is the most physiologically significant inhibitor of the TF-FVIIa- triggered coagulation pathway; nevertheless, very few mechanisms/factors that could regulate the natural expression of TFPI have been identified so far. Here we describe androgen treatment of EC as a novel way to preserve and/or enhance a healthy vascular function, particularly related to the regulation of TFPI-dependent anticoagulant function of the endothelium. Our hypothesis is that a yet uncharacterized protein encoded by C6orf105 is a novel regulator of TFPI expression and function in EC, both in native conditions and during androgen stimulation. “In silico” data mining using global meta-analysis of publicly available NCBI's Gene Expression Omnibus 2-channel human microarray datasets identified C6orf105 as highly co-expressed with TFPI and following a parallel co-regulation. The uncharacterized protein has 230-aa, Mr ∼27 kDa, 5–6 predicted transmembrane domains and has sequence similarities with members of the androgen-inducible genes family. We tentatively named it TFPI-Regulating Factor (TFPI-RF). Real-time qPCR and western blot confirmed robust expression of TFPI-RF in EC in culture (HUVEC and EA.hy926 hybrid cell line). By immunofluorescence (IMF) TFPI-RF appears both on the cell surface and intracellularly co-localizing with TFPI and caveolin-1 (cav-1). Post-transcriptional (siRNA) down-regulation of TFPI-RF decreased TFPI, both as protein (∼2-times) and as anticoagulant activity (∼3-fold), apparently by reducing the co-localization of the TF-FVIIa-FXa-TFPI complex with cav-1. Over-expression of TFPI-RF in HUVEC and EA.hy926 led to enhanced co-localization of TFPI-RF with TFPI, and increased TFPI mRNA and anticoagulant activity (∼2-times). Western blot of cellular fractions after extraction with Triton X-114 and temperature-induced phase separation revealed the presence of TFPI and TFPI-RF in detergent-insoluble fractions, which suggests predominant lipid raft association. IMF illustrates TFPI-RF co-clustering with TFPI and cav-1 or GM1 (raft marker) in live EC incubated with anti-TFPI antibody or Cholera Toxin-B, respectively. The effect of androgens was studied by incubating EC with 30 nM dehydrotestosterone (DHT) or equivalent testosterone-BSA (cell-impermeable). 1-h incubation led to 2-times enhanced TFPI activity, increased co-localization of the quaternary complex with cav-1 and TFPI-RF, and enhanced exposure of TFPI and TFPI-RF on the cell surface. 24-h treatment with DHT up-regulates the expression of both TFPI (2-fold) and TFPI-RF (3-fold), as well as the TFPI inhibitory activity against FXa. DHT failed to enhance TFPI activity in TFPI-RF siRNA EC. Our results reveal a novel mechanism of up-regulation of the anticoagulant activity of endogenous TFPI in response to physiological levels of androgen. While the precise role of androgens in the ageing process is unclear, it is believed that androgen replacement could have beneficial influence on the declining functions in the elderly. Our data could expand on the effects of androgens on the haemostatic function of the endothelium and discover new roles for novel proteins like C6orf105/TFPI-RF in enhancing the endothelial anticoagulant function. These may open possibilities to manipulate the cellular endogenous TFPI and/or other intrinsic factors to counteract pro-thrombotic states associated with CVD, DVT/VTE, sepsis and cancer. Disclosures:No relevant conflicts of interest to declare.

The Mir-23a∼Mir-27a∼Mir-24 Cluster Acts as a Tumor Suppressor In Leukemias by Post-Transcriptional Regulation of 14-3-3 Proteins

AbstractAbstract 3145We and others have found that microRNAs (miRs) play major roles in normal hematopoietic differentiation, evidenced by the discovery of a small set of hematopoietic stem-progenitor cell (HSPC)-expressed miRs (HE-miRs) highly expressed in normal human CD34+ cells, which post-transcriptionally regulate specific mRNAs involved in hematopoiesis. Microarray data indicated that 1 of these HE-miRs, miR-27a (transcribed as part of the miR-23a cluster consisting of 3 coordinately transcribed miRs: miR-23a, miR-27a, miR-24-2) is absent or expressed at much lower levels in several human acute leukemias, as compared to normal human HSPCs. We further investigated the expression of miR-27a in acute leukemias, and confirmed via qRT-PCR that there is ≥ 2-fold decreased miR-27a expression in 3 of 6 acute myeloid leukemia (AML), 4 of 5 B precursor acute lymphoid leukemia (ALL), and 3 of 3 T cell ALL cell lines tested (10 of 14 cell lines total). Additionally, 5 of 7 primary B precursor ALL and 5 of 5 primary T cell ALL samples had decreased miR-27a expression compared to normal HSPCs. Over-expression of miR-27a in K562 cells resulted in 4-fold decreased proliferation and 2-fold increased apoptosis compared to control cells. Similarly, over-expression of miR-27a in 3 other acute leukemia cell lines (TF1, HL60, and REH) yielded a significant increase in the percentage of AnnexinV+/7AAD- cells compared to control cells (TF1 = 21.3% vs. 12.0%; HL60 = 19% vs. 4.5%; REH = 21.5% vs. 4.7%). Members of the 14-3-3 protein family act as oncogenes and support cell survival by interacting with and negatively regulating pro-apoptotic proteins such as Bax and Bad. The 14-3-3β isoform (YWHAB) is a predicted miR-27a target gene, based on transcriptome analysis of normal HSPCs, and miR prediction algorithms revealed 2 predicted miR-27a binding sites in its 3'UTR. In addition, there are 5 predicted miR-24 binding sites in the 3'UTR and coding region of YWHAB, and 4 other 14-3-3 isoforms have multiple predicted miR-23a cluster member binding sites in their mRNA transcripts: 14-3-3θ (YWHAQ, 1 miR-27a site), 14-3-3γ (YWHAG, 2 miR-23a, 5 miR-27a and 3 miR-24 sites), 14-3-3ζ (YWHAZ, 3 miR-27a and 5 miR-24 sites), and 14-3-3ε (YWHAE, 1 miR-27a and 1 miR-24 site). The expression levels of the other 2 cluster members, miR-23a and miR-24, are also decreased in human acute leukemia cell lines and patent samples, similar to miR-27a expression in the same leukemia cases. We have confirmed via luciferase reporter binding assays and Western blot analyses that both miR-27a and miR-24 specifically regulate 14-3-3β protein expression levels, while miR-27a specifically regulates 14-3-3θ. Moreover, combined miR-27a and miR-24 over-expression decreased 14-3-3ε protein expression on Western blots. Since knockdown of 14-3-3 family proteins is established to result in apoptosis in hematopoietic cells including K562, post-transcriptional downregulation of 14-3-3 proteins by miR-27a, miR-23a and miR-24 contributes to the miR-27a-induced apoptosis in leukemias. Thus, miR-27a acts as a traditional tumor suppressor gene and members of the miR-23a cluster cooperate to regulate oncogenic 14-3-3 isoforms, and consequently, cell survival, in human acute leukemias. Disclosures:No relevant conflicts of interest to declare.

Halofuginone Induces Post-Transcriptional Down-Regulation of Cyclin D1, Cell Cycle Arrest and Apoptosis In Mantle Cell Lymphoma Cells through Activation of Integrated Stress Response Pathways

AbstractAbstract 773Mantle cell lymphoma (MCL) remains an incurable disease and has the worst outcome among B-cell lymphomas. Patients generally have a good response to first line treatment but most relapse and tend to have shorter responses or resistant disease. Thus, novel treatment strategies capable of providing and sustaining durable responses are clearly needed. The translocation t(11;14), a hallmark of MCL, leads to cyclin D1 overexpression and is invariably accompanied by different secondary genetic lesions that collaborate for lymphomagenesis. In a previous study, we found that several genes related to the AKT, WNT and TGFβ signaling pathways were aberrantly expressed in MCL. The role of the AKT and WNT pathways in MCL pathogenesis has been well established by other groups, but little is known about the role of the TGFβ pathway. To address this issue, we tested whether halofuginone, a small molecule with recognized anti-TGFβ and antifibrotic activity, would have cytotoxic effect against a panel of MCL cell lines. We found that halofuginone at nanomolar levels had significant cytotoxic activity against MCL cell lines as measured by the MTT assay. The IC50‘s for Mino and HBL-2 cell lines were 30 and 61 ng/mL at 48h, respectively, with IC50‘s for Jeko-1, JVM-2 and Granta-519 falling in between. Halofuginone induced apoptosis in Mino and HBL-2 cells in a time- and concentration-dependent fashion, as evidenced by annexin V/7-AAD staining by flow cytometry and electron microscopy studies. However, halofuginone failed to inhibit SMAD2 phosphorylation induced by recombinant TGFβ1 in Mino and HBL-2 cells, as shown by Western blot analysis, and co-treatment experiments with TGFβ1 failed to show antagonism, suggesting that the effect of halofuginone in MCL is not mediated by TGFβ inhibition. Cell cycle analysis of Mino and HBL-2 cells exposed to halofuginone revealed time- and concentration-dependent accumulation in G1 (83% of Mino cells at G1 upon exposure to 50 ng/mL for 24h vs. 48% in untreated Mino cells), and immunocytochemical analysis showed that this effect was accompanied by striking down-regulation of cyclin D1 protein levels starting as early as 3h after exposure to halofuginone, a finding that was reproduced in primary MCL cells. Real-time RT-PCR experiments, however, revealed up-regulation of cyclin D1 mRNA levels by halofuginone over time, suggesting a post-transcriptional mechanism for the observed down-regulation of cyclin D1 protein levels. Western blot analysis of Mino and HBL-2 cells exposed to halofuginone for 24h showed a concentration-dependent phosphorylation of GCN2, PERK and EIF2α, and up-regulation of ATF4. These findings point to an activation of integrated stress response pathways (amino acid starvation response and endoplasmic reticulum stress response) that causes a general shutdown in protein synthesis and explain, at least partially, the down-regulation in cyclin D1 levels. To further characterize the proteins targeted by halofuginone in MCL we employed a proteomic profiling approach in which differentially expressed proteins were revealed by label-free liquid chromatography tandem mass spectrometry (LC-MSE) analysis on a nanoAcquity system coupled to a Synapt MS Q-Tof mass spectrometer. A comprehensive catalogue representing 147 proteins was generated from this analysis and we found that several members of the heat shock protein family are up-regulated in Mino cells exposed to 100 ng/mL of halofuginone for 14h, the relevance of which is currently under investigation. Together, our data demonstrate that halofuginone at nanomolar levels has significant antiproliferative and cytotoxic effects in MCL cells that are induced by the activation of integrated stress response pathways. More importantly, our study provides a rationale for exploring the clinical activity of this oral agent in patients with MCL. Disclosures:No relevant conflicts of interest to declare.

Reduction of transforming growth factor‐β type II receptor is caused by the enhanced ubiquitin‐dependent degradation in human renal cell carcinoma

Although dysregulation of transforming growth factor-beta (TGF-beta) signaling is implicated in renal carcinogenesis, its precise mechanism is unknown in renal cell carcinoma (RCC). In our study, we investigated Smad-mediated TGF-beta signaling pathway and its regulatory mechanisms in surgical samples from patients with RCC. We found that immunoreactivity for nuclear phosphorylated Smad2 was significantly decreased in RCC compared to normal renal tissues, thereby TGF-beta signaling was suggested to be attenuated in RCC tissues. In accordance with the result, transcriptional downregulation of Smad4 and post-transcriptional downregulation of TGF-beta type II receptor (TbetaR-II) were frequently found in RCC tissues compared to normal renal tissues. Next, to clarify the reason why the protein level of TbetaR-II was decreased in RCC, we investigated the activities of degradation and ubiquitination of TbetaR-II. We found that both proteasome-mediated degradation and ubiquitination of TbetaR-II were markedly enhanced in RCC tissues. Moreover, we found that the level of Smad-ubiquitination regulatory factor 2 (Smurf2), the E3 ligase for TbetaR-II, was increased in RCC tissues of the patients with higher clinical stages compared to the normal tissues and was inversely correlated with the level of TbetaR-II. Our results suggest that the low TbetaR-II protein level is due to augmented ubiquitin-dependent degradation via Smurf2 and might be involved in the attenuation of TGF-beta signaling pathway in RCC.

Fungi Unearthed: Transcripts Encoding Lignocellulolytic and Chitinolytic Enzymes in Forest Soil

BackgroundFungi are the main organisms responsible for the degradation of biopolymers such as lignin, cellulose, hemicellulose, and chitin in forest ecosystems. Soil surveys largely target fungal diversity, paying less attention to fungal activity.Methodology/Principal FindingsHere we have focused on the organic horizon of a hardwood forest dominated by sugar maple that spreads widely across Eastern North America. The sampling site included three plots receiving normal atmospheric nitrogen deposition and three that received an extra 3 g nitrogen m−2 y−1 in form of sodium nitrate pellets since 1994, which led to increased accumulation of organic matter in the soil. Our aim was to assess, in samples taken from all six plots, transcript-level expression of fungal genes encoding lignocellulolytic and chitinolytic enzymes. For this we collected RNA from the forest soil, reverse-transcribed it, and amplified cDNAs of interest, using both published primer pairs as well as 23 newly developed ones. We thus detected transcript-level expression of 234 genes putatively encoding 26 different groups of fungal enzymes, notably major ligninolytic and diverse aromatic-oxidizing enzymes, various cellulose- and hemicellulose-degrading glycoside hydrolases and carbohydrate esterases, enzymes involved in chitin breakdown, N-acetylglucosamine metabolism, and cell wall degradation. Among the genes identified, 125 are homologous to known ascomycete genes and 105 to basidiomycete genes. Transcripts corresponding to all 26 enzyme groups were detected in both control and nitrogen-supplemented plots.Conclusions/SignificanceMany of these enzyme groups are known to be important in soil turnover processes, but the contribution of some is probably underestimated. Our data highlight the importance of ascomycetes, as well as basidiomycetes, in important biogeochemical cycles. In the nitrogen-supplemented plots, we have detected no transcript-level gap likely to explain the observed increased carbon storage, which is more likely due to community changes and perhaps transcriptional and/or post-transcriptional down-regulation of relevant genes.

BIK (Bcl2-Interacting Killer) CpG methylation status as a potential predictive biomarker of relapsed/refractory multiple myeloma disease.

8118 Background: BIK (bcl2-interacting killer) is the founding member of the BH3-only family proapoptotic proteins. Genomic deletions, epigenetic silencing or post-transcriptional downregulation of BIK by the proteasome and induction of apoptosis upon BIK re-expression reinforce the notion that BIK is a tumor suppressor. Although it has been shown that BIK is a target of epigenetic silencing in cell lines, no studies exist regarding the CpG methylation status of BIK in MM patients. Methods: Bone marrow aspirate samples from MM patients were obtained at diagnosis. Methylation-specific PCR (MSP) was employed to study methylation in the BIK CpG island. Genomic DNA was isolated and bisulphite modification performed using commercially available kits. Control methylated and unmethylated genomic DNAs were included in each experiment. Logistic regression analysis was used to measure the association between gene methylation and age, ISS stage, ECOG performance status, extramedullary disease, bone disease, anemia (Hb <10 g/dl), serum albumin and beta 2 microglobulin levels and relapsed/refractory disease. Results: Methylation in the BIK CpG island was studied in 40 MM patients (21 male, 19 female, mean age 66). Methylation of the BIK CpG island was found in 16 patients (40%) and men were more likely to be methylated (OR=3.08, p=0.01) than women. No correlation was found between BIK CpG methylation and age, ECOG performance status, ISS staging, anemia, beta 2 microglobulin levels, albumin levels, and overall survival. Patients methylated for BIK had a 2-fold tendency to have bone disease (p=0.35) and a 3-fold tendency to have extramedullary disease (p=0.14). Notably, patients with methylated BIK had a significantly higher risk of relapsed/refractory disease (OR=5.4, p=0.033). Conclusions: To the best of our knowledge, this is the first demonstration of aberrant methylation in the BIK CpG island in MM patients. BIK CpG methylation in this series of patients was found to be strongly associated with relapsed/refractory disease. Patients with refractory/relapsed disease could possibly benefit from agents enhancing BIK expression, such as proteasome inhibitors. No significant financial relationships to disclose.

Distinct molecular mechanisms leading to deficient expression of ER-resident aminopeptidases in melanoma.

Immune surveillance of tumour cells by CD8(+) cytotoxic T cells plays a key role in the establishment and control of an anti-tumour response. This process requires the generation of antigenic peptides, which are largely produced by the proteasome in combination with other proteases located in either the cytoplasm and/or the endoplasmic reticulum (ER). The ER-resident aminopeptidases ERAP1 and ERAP2 trim or even destroy HLA class I-binding peptides thereby shaping the peptide repertoire presented for T cell recognition. So far there exists limited information about the expression pattern of ERAP1 and/or ERAP2 in human tumours of distinct histotypes. Therefore, the expression profiles and modes of regulation of both aminopeptidases were determined in a large series of melanoma cell lines. A heterogeneous expression ranging from high to reduced or even total loss of ERAP1 and/or ERAP2 mRNA and/or protein expression was detected, which often could be induced/upregulated by interferon-gamma treatment. The observed altered ERAP1 and/or ERAP2 expression and activity levels were either mediated by sequence alterations affecting the promoter or enzymatic activities, leading to either transcriptional and/or post-transcriptional downregulation mechanisms or limited or excessive processing activities, which both might have an impact on the antigenic peptide repertoire presented on HLA class I molecules.

Regulation of Emx2 Expression by Antisense Transcripts in Murine Cortico-Cerebral Precursors

Background Emx2 encodes for a transcription factor expressed in the embryonic intermediate mesoderm and central nervous system (CNS). It is implicated in several aspects of cerebral cortex development, including morphogenetic field specification, arealization, precursor proliferation and lamination. Four Emx2-associated antisense transcripts have been found in the urogenital system; one of them, Emx2OS, has been also detected in the adult brain. Until now, however, nothing is known about expression and function of Emx2OS in the developing CNS.Methodology/Principal FindingsBy quantitative RT-PCR and in situ hybridization, we reconstructed the Emx2OS expression profile in the embryonic CNS, paying special attention to the developing cerebral cortex. Emx2OS was observed in a number of CNS structures expressing also Emx2. Within the cortex, Emx2OS was detectable in periventricular precursors, expressing the sense transcript, and peaked in newly born post-mitotic neurons not expressing such transcript. By integrating lentiviral gene delivery, RNAi, TetON technology, morpholino-mediated gene knock-down, drug-induced perturbation of gene expression, and quantitative RT-PCR, we addressed possible roles of Ex2 antisense RNA in Emx2 regulation, in primary CNS precursor cultures. We found that, in both cortical precursors and their neuronal progenies, Emx2 antisense RNA contributes to post-transcriptional down-regulation of its sense partner, possibly by a Dicer-promoted mechanism. The same RNA, when delivered to rhombo-spinal precursors, stimulates ectopic expression of Emx2, whereas Emx2 knock-out dramatically impairs Emx2OS transcription. This suggests that, within the developing CNS, a reciprocal Emx2/Emx2OS regulatory loop may normally sustain transcription at the Emx2 locus.Conclusions/SignificanceThis study shows that antisense transcripts may contribute to developmental regulation of a key transcription factor gene implicated in CNS patterning, possibly by complex and multilevel mechanisms. The activation of Emx2 by a short antisense transcript may be a prototype of a method for overexpressing single specific genes, without introducing additional copies of them into the genome.

Suppression of PLCβ2 by Endotoxin Plays a Role in the Adenosine A 2A Receptor-Mediated Switch of Macrophages from an Inflammatory to an Angiogenic Phenotype

Toll-like receptor (TLR) 2, 4, 7, and 9 agonists, together with adenosine A(2A) receptor (A(2A)R) agonists, switch macrophages from an inflammatory (M1) to an angiogenic (M2-like) phenotype. This switch involves induction of A(2A)Rs by TLR agonists, down-regulation of tumor necrosis factor alpha (TNFalpha) and interleukin-12, and up-regulation of vascular endothelial growth factor (VEGF) and interleukin-10 expression. We show here that the TLR4 agonist lipopolysaccharide (LPS) induces rapid and specific post-transcriptional down-regulation of phospholipase C(PLC)beta1 and beta2 expression in macrophages by de-stabilizing their mRNAs. The PLCbeta inhibitor U73122 down-regulates TNFalpha expression by macrophages, and in the presence of A(2A)R agonists, up-regulates VEGF, mimicking the synergistic action of LPS with A(2A)R agonists. Selective down-regulation of PLCbeta2, but not PLCbeta1, using small-interfering RNA resulted in increased VEGF expression in response to A(2A)R agonists, but did not suppress TNFalpha expression. Macrophages from PLCbeta2(-/-) mice also expressed increased VEGF in response to A(2A)R agonists. LPS-mediated suppression of PLCbeta1 and beta2 is MyD88-dependent. In a model of endotoxic shock, LPS (35 microg/mouse, i.p.) suppressed PLCbeta1 and beta2 expression in spleen, liver, and lung of wild-type but not MyD88(-/-) mice. These studies indicate that LPS suppresses PLCbeta1 and beta2 expression in macrophages in vitro and in several tissues in vivo. These results suggest that suppression of PLCbeta2 plays an important role in switching M1 macrophages into an M2-like state.

Insulin Enhances the Biogenesis of Nuclear Sterol Regulatory Element-binding Protein (SREBP)-1c by Posttranscriptional Down-regulation of Insig-2A and Its Dissociation from SREBP Cleavage-activating Protein (SCAP)·SREBP-1c Complex

The regulation of lipid homeostasis by insulin is mediated in part by the enhanced transcription of the gene encoding sterol regulatory element-binding protein-1c (SREBP-1c). The nascent SREBP-1c is embedded in the endoplasmic reticulum (ER) and must be transported to the Golgi where two sequential cleavages generate its NH(2)-terminal fragment, nSREBP-1c. We have shown recently that in primary cultures of rat hepatocytes, insulin rapidly and selectively stimulates proteolytic processing of the nascent SREBP-1c by enhancing the affinity of the SREBP cleavage-activating protein (SCAP).SREBP-1c complex for coatomer protein complex II (COPII) vesicles. The SCAP.SREBP complex is retained in the ER by Insig proteins. We report here that insulin persistently stimulates controlled proteolysis of the nascent SREBP-1c by selectively reducing the level of Insig-2a protein via accelerated degradation of its cognate mRNA. Insulin enhanced the rate of turnover of Insig-2a mRNA via its 3'-untranslated region. Insulin-induced depletion of Insig-2a promotes association of the SCAP.SREBP-1c complex with COPII vesicles and subsequent migration to the Golgi where site-1 and site-2 proteases process the nascent SREBP-1c. Consistent with this mechanism, experimental knockdown of Insig-2a expression with small interfering RNA mimicked insulin-induced proteolysis of the nascent SREBP-1c, whereas exogenous expression of Insig-2a in hepatocytes led to reduced intramembrane proteolysis of the newly synthesized SREBP-1c. The action of insulin on the processing of the nascent SREBP-1c via Insig-2a was highly selective, as proteolysis of the newly synthesized SREBP-2 remained unchanged under identical conditions. On the basis of these data, we propose that the stimulation of SREBP-1c processing by insulin is mediated by a selective depletion of Insig-2a protein by promoting decay of its cognate mRNA. Thus, insulin-induced reduction in Insig-2a protein leads to an enhanced export of the SCAP.SREBP-1c complex from ER to the Golgi.

Dual Functions of Rift Valley Fever Virus NSs Protein: Inhibition of Host mRNA Transcription and Post‐transcriptional Downregulation of Protein Kinase PKR

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis, or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-beta mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase (PKR), to prevent phosphorylation of eIF2alpha and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts.

Differential regulation of the serotonin 1 A transcriptional modulators five prime repressor element under dual repression-1 and nuclear-deformed epidermal autoregulatory factor by chronic stress

Chronic stress is known to affect brain areas involved in learning and emotional responses. These changes, thought to be related to the development of cognitive deficits are evident in major depressive disorder and other stress-related pathophysiologies. The serotonin-related transcription factors (Freud-1/CC2D1A; five prime repressor element under dual repression/coiled-coil C2 domain 1a, and NUDR/Deaf-1; nuclear-deformed epidermal autoregulatory factor) are two important regulators of the 5-HT1A receptor. Using Western blotting and quantitative real-time polymerase chain reaction (qPCR) we examined the expression of mRNA and proteins for Freud-1, NUDR, and the 5-HT1A receptor in the prefrontal cortex (PFC) of male rats exposed to chronic restraint stress (CRS; 6 h/day for 21 days). After 21 days of CRS, significant reductions in both Freud-1 mRNA and protein were observed in the PFC (36.8% and 32%, respectively; P<0.001), while the levels of both NUDR protein and mRNA did not change significantly. Consistent with reduced Freud-1 protein, 5-HT1A receptor mRNA levels were equally upregulated in the PFC, while protein levels actually declined, suggesting post-transcriptional receptor downregulation. The data suggest that CRS produces distinct alterations in the serotonin system specifically altering Freud-1 and the 5-HT1A receptor in the PFC of the male rat while having no effect on NUDR. These results point to the importance of understanding the mechanism for the differential regulation of Freud-1 and NUDR in the PFC as a basis for understanding the related effects of chronic stress on the serotonin system (serotonin-related transcription factors) and stress-related disorders like depression.