GW3965 treatment up-regulates ABCA1 expression and its cholesterol efflux function.

<p>(A) Cell toxicity of GW3965. Huh7.5 cells were cultured in the presence of indicated concentrations of the drug for 24 h. The luminescent signal is expressed in luminescence units (RLU). (B) Up-regulation of ABCA1 mRNA expression by GW3695 treatment. Huh7.5 cells were treated for 24 h with 1 µM GW3695 or drug solvent (DMSO). Then ABCA1 mRNA was determined by qRT-PCR. (C) ABCA1 protein production in drug-stimulated Huh7.5 cells. Cells were treated for 24 h with 1 µM GW3965 and analysed by Western blot (shown in the insert). Protein content in the ABCA1 band (220 kDA) in GW3965-(GW), and DMSO-(solv) treated cells was quantified relative to the calnexin band using the Odyssey Infrared Imaging System. (D) GW3965 stimulation promotes ABCA1-mediated cholesterol efflux to ApoA1. Huh7.5 cells were labelled with [³H] cholesterol then incubated with GW3965 or drug solvent. ABCA1-dependent [³H] cholesterol efflux was assayed by comparing cell-associated and free radioactivity. (E) Kinetics of ABCA1 gene expression following stimulation of cells with GW3965. Huh7.5 cells were treated with 1 µM GW3965 for the indicated time and ABCA1 mRNA was determined by qRT-PCR. Results were expressed as relative values compared to ABCA1 expression in cells treated with drug solvent. (F) Kinetics of cholesterol efflux in cells stimulated with GW3965. Huh7.5 cells were labelled with [³H] cholesterol for 24 h, and incubated for an additional 16 h with 1 μM GW3965 or drug solvent. ABCA1-dependent [³H] cholesterol efflux was assayed in the presence of ApoA1 and either GW3965 or solvent for the indicated period of time.</p

<i>NCYM</i>, a <i>Cis</i>-Antisense Gene of <i>MYCN</i>, Encodes a <i>De Novo</i> Evolved Protein That Inhibits GSK3β Resulting in the Stabilization of MYCN in Human Neuroblastomas

<div><p>The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, <i>de novo</i> gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that <i>NCYM</i>, a <i>cis</i>-antisense gene of the <i>MYCN</i> oncogene, initially thought to be a large non-coding RNA, encodes a <i>de novo</i> evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The <i>NCYM</i> gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, <i>NCYM</i> is 100% co-amplified and co-expressed with <i>MYCN</i>, and <i>NCYM</i> mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both <i>NCYM</i> and <i>MYCN</i> mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to <i>MYCN</i> transgenic mice, neuroblastomas in <i>MYCN/NCYM</i> double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with <i>MYCN</i> amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the <i>MYCN</i>/<i>NCYM</i> double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both <i>in vitro</i> and <i>in vivo</i>. Furthermore, the survival of <i>MYCN</i> transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in <i>MYCN/NCYM</i> double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first <i>de novo</i> evolved protein known to act as an oncopromoting factor in human cancer, and suggest that <i>de novo</i> evolved proteins may functionally characterize human disease.</p></div

Up-regulation of ABCA1 inhibits HCV cell entry.

<p>The effect of GW3965 on the HCV cell cycle was analysed by adding the drug at different time points. A flow-chart is depicted in the upper panel of the graph. RNA in Huh7.5 cells infected in the presence of DMSO is shown in (a); that in cells pre-treated for 24 h with 1 µM GW3965 and infected in the presence of the drug are shown in (b), (c) and (d); results for cells treated with GW3965 during virus inoculation without pre-treatment are shown in (e); those of assays where the drug was added at 2 h, 4 h, or 6 h post-infection are presented in (f), (g) and (h) respectively. For each experiment cells were incubated for the indicated time period after infection (IV). The efficiency of infection was expressed as intracellular HCV RNA measured by qRT-PCR as a per cent of the control (a).</p

Analysis of HCV particles secreted from cells that over-express ABCA1.

<p>Physical properties of the nascent virus particles produced in cells stimulated or not with GW3965 were analysed by centrifugation in iodixanol gradient. Huh7.5 cells were pre-incubated with solvent (panel A) or 1 µM GW3965 (panel B) and the drug was maintained until 72 h post-infection when cell supernatants were collected, concentrated and subjected to gradient centrifugation. HCV RNA in gradient fractions was quantified by qRT-PCR and core antigen, ApoB and ApoE by ELISA assays.</p

GW3695 treatment modulates expression of genes involved in lipid metabolism.

<p>Huh7.5 cells were treated with 1 µM GW3695. Total RNA was extracted from cells and the mRNA levels corresponding to several genes regulating lipoprotein metabolism: ABCA1, ABCG1, nuclear LXRα and LXRβ receptors, a sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS) and phospholipid transfer protein (PLTP), CD36 and ApoA1 were determined by qRT-PCR. The results were normalized to housekeeping genes and compared to the levels of corresponding mRNAs in solvent-treated cells.</p

Over-expression of ABCA1 inhibits HCV infection of primary human hepatocytes and human liver slices.

<p>(A–B) Inhibition of HCV infection of primary human hepatocytes. (A) Primary human hepatocytes were treated with 2–10 μM GW3965 (non-toxic concentrations for cells) or with drug solvent, prior to HCV infection. Twenty-four hours post-infection, ABCA1 mRNA was determined by qRT-PCR and expressed in arbitrary units, taking into account ABCA1 levels in liver cells pre-treated with the drug. (B) GW3965-treated and solvent-treated primary human hepatocytes were inoculated with HCV. After 24 h, intracellular HCV RNA was quantified by qRT-qPCR. The efficiency of infection in drug pre-treated cells was expressed as the percentage of infection compared to solvent-treated cells. (C–D) ABCA1 over-expression inhibits HCV infection of human liver slices. Human liver slices were cultured for 24 h, treated with 5 or 10 μM GW3965 or with DMSO before infection with HCVcc. At 24 h post-infection, total RNA was extracted and ABCA1 mRNA (C) and HCV RNA (D) were quantified by corresponding qRT-PCR assays and expressed as the percentage of RNA compared to the values obtained for solvent-treated cells.</p

Stimulation of ABCA1 inhibits HCV infection.

<p>(A) Reduction of intracellular HCV RNA levels in cells that over-express ABCA1. Huh7.5 cells were pre-treated with 1 µM GW3965 then infected with HCV. Cells were grown for a further 24 h, total RNA was extracted and intracellular HCV RNA was determined by qRT-PCR. Results are expressed as the percentage of HCV RNA relative to that in cells treated with drug solvent prior to infection. (B) Decrease of HCV RNA levels in the supernatant collected from drug-stimulated cells. Huh7.5 cells were pre-treated with 1 µM GW3965 then infected with HCV. After a further 72 h, HCV-RNA in the culture medium was determined by qRT-PCR. Results are expressed as the percentage of HCV RNA secreted from drug-treated cells compared to solvent-treated cells. (C) Effect of GW3965 treatment on long-term HCV infection. Huh 7.5 cells were pre-treated with 1 µM GW3965, infected with HCV and grown for up to 7 days in the presence of the drug. ABCA1 mRNA was determined by qRT-PCR every 24 h and results are expressed as a fold-increase of ABCA1 mRNA compared to solvent-treated cells (grey bars). HCV RNA in the cell supernatant was measured at the same time points by qRT-PCR (line curves for GW3965 treated [filled triangles] or control [filled squares] cells) and is expressed in International Units (IU).</p

Functional interaction between NCYM and MYCN.

<p>(A) Relative mRNA levels of <i>NCYM</i> in SK-N-AS <i>MYCN</i> single copy human neuroblastoma cells transfected with MYCN expression vector. mRNA levels were measured by qRT-PCR with <i>β-actin</i> as an internal control. (B) Relative mRNA levels of <i>NCYM</i> (left panel) or <i>MYCN</i> (right panel) upon depletion of MYCN in CHP134 human <i>MYCN</i>-amplified neuroblastoma cells. (C) MYCN enhances <i>NCYM</i> promoter activity. Human neuroblastoma SK-N-AS cells were transfected with increasing amounts of MYCN expression plasmid (0, 200, 300 ng) and their luciferase activity was measured. (D) Western blots showing NCYM overexpression induces MYCN protein in Neuro 2a mouse neuroblastoma cells (left panel). <i>MYCN</i> mRNA expression in mouse neuroblastoma Neuro 2a cells transfected with increasing amounts of NCYM expression vector measured by qRT-PCR (right panel). (E) Western blots showing NCYM knockdown decreases MYCN protein in CHP134 cells (left panel). <i>MYCN</i> mRNA expression in NCYM knockdown CHP134 cells as measured by qRT-PCR (right panel). (F, G) Co-immunoprecipitation of endogenous NCYM with endogenous MYCN and GSK3β. (H) GST-pulldown assay. Purified NCYM proteins were pulled down with GST-fused GSK3β and MYCN. (I) <i>In vitro</i> kinase assay. Radiolabeled ATP was used for the second reaction with GSK3β together with the indicated amount of NCYM or GST. The amount of phosphorylated MYCN was quantified using standard autoradiography. The total amount of the MYCN was quantified by using an Oriole Fluorescent Gel stain.</p

<i>NCYM</i> encodes a <i>de novo</i> evolved protein in humans.

<p>(A) Gene structure of the human <i>MYCN</i>/<i>NCYM</i> locus. (B) Alignment of the possible amino acid sequences of NCYM in the human and primate genomes, where the ORF of the primate genes begins at the same position as the human start codon. Red text indicates amino acid differences compared with human NCYM. (C) Change in protein features along the lineage shown. CA indicates common ancestor. L indicates the sequence length of amino acids before the first terminal codon. Asterisk indicates statistical significance (**<i>P</i><0.001, *<i>P</i><0.05). <i>K</i>_a and <i>K</i>_s indicate the rate of non-synonymous changes and synonymous changes, respectively. (D–G) The protein expression of NCYM and MYCN in human primary neuroblastomas (D, E) and normal human cerebrum (F, G). Scale bars, 100 µm (D, E) and 50 µm (F, G). Sections of neuroblastomas with <i>MYCN</i> amplification and those of normal human cerebrum were stained with anti-NCYM (D, F) or anti-MYCN (E, G) antibodies.</p

<i>NCYM</i> expression is associated with poor prognosis in human neuroblastoma.

<p>(A) <i>NCYM</i> mRNA expression correlates with that of <i>MYCN</i> in human primary neuroblastomas (n = 106, Rs. = 0.686, <i>P</i> = 4.69×10⁻¹⁶). (B) <i>NCYM</i> mRNA expression correlates with that of <i>MYCN</i> in human primary neuroblastomas with <i>MYCN</i> single copy (n = 86, Rs. = 0.695, <i>P</i> = 1.11×10⁻¹³). The mRNA expression of <i>NCYM</i> and <i>MYCN</i> was detected by qRT-PCR and normalized using <i>GAPDH</i>. (C) Kaplan–Meier survival curves (n = 106, <i>P</i> = 3.70×10⁻⁵, log-rank test). The expression levels of <i>NCYM</i> were designated high (n = 13, closed circle) or low (n = 93, open circle) based on the respective average expression. (D) Kaplan–Meier survival curves. The expression levels of <i>MYCN</i> were designated high (n = 15, closed circle) or low (n = 91, open circle) based on the respective average expression. High <i>MYCN</i> mRNA expression was significantly correlated with poor prognosis (n = 106, <i>P</i> = 2.31×10⁻⁵, log-rank test).</p