<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

<i>MYCN</i>/<i>NCYM</i> tumors show drug resistance to a PI3K/mTOR-dual inhibitor.

<p>(A) Western blots of <i>MYCN</i> (M2–M6) and <i>MYCN</i>/<i>NCYM</i> (M7–M11) mouse tumors for NCYM, phospho-GSK3β (S9), GSK3β, β-catenin, and MYCN, phospho-S6K (T389), S6K, phospho-AKT (S473), phospho-AKT (T308), and AKT. Actin was used as loading control. (B) NCYM binds to GSK3β <i>in vivo</i>. Tumors developed in <i>MYCN/NCYM</i> transgenic mice (M12) were immunoprecipitated with control IgG or NCYM antibodies. GSK3β was co-immunoprecipitated with a NCYM antibody. (C, D) Kaplan–Meier survival analysis of <i>MYCN</i> mice (panel C, <i>P</i><0.01, log-rank test) or <i>MYCN</i>/<i>NCYM</i> mice (panel D, <i>P</i> = 0.648, log-rank test). Treatment with NVP-BEZ235 (35 mg/kg; open circles) or vehicle (PEG300; closed circles). NVP-BEZ235, <i>MYCN</i> transgenic mice n = 7, <i>MYCN</i>/<i>NCYM</i> transgenic mice n = 11; PEG300, <i>MYCN</i> transgenic mice n = 5, <i>MYCN</i>/<i>NCYM</i> transgenic mice n = 7.</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

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