Abstract 4085: Bidirectional noncoding RNAs mediate rRNA processing and cell death in human lung adenocarcinoma cells
Authors: Hwang Christopher J. , Fields Janet R. , Anderson Lucy M. , Shiao Yih-Horng 
Published: February 2011

Abstract
Increase in ribosome biogenesis is a characteristic of proliferating cells and may be causative in tumorigenesis. Significant increases in basal levels of several primary precursor transcripts during ribosomal RNA (rRNA) processing and of a cis antisense noncoding rRNA (AS-ncrRNA) near the main transcription start site were detected in human lung adenocarcinoma cells (H441 and A549) compared with non-transformed peripheral lung epithelial cells (HPL). A cis sense noncoding rRNA (S-ncrRNA) was also discovered but its levels showed cell-specific changes. It was hypothesized that a high level of the AS-ncrRNA is required to maintain the tumor phenotype. The functions of the AS-ncrRNA and S-ncrRNA were assessed using complementary oligonucleotides, termed −86S and −84AS (negative numbers correspond to the target locations in reference to the primary transcription start site) and intended for inhibiting the above targets, respectively. The levels of precursor rRNAs and both ncrRNAs in cells transfected with the −86S and −84AS oligonucleotides were measured using reverse transcription and quantitative polymerase chain reaction. Cell phenotypes and apoptotic cell death were also examined. A two-hit treatment with −86S 24 hrs apart induced cell detachment predominantly in H441 and A549 cancer cells. Detached cancer cells showed several hundred- to a thousand-fold upregulation of S-ncrRNA over the vehicle-treated controls while the S-nrcRNA in rare floating HPL cells was only increased by about 20 fold. Precursor rRNAs and AS-ncrRNA were also upregulated but to a lesser extent after −86S treatment in both adherent and detached cells. For the −84AS treatment, detached cells were fewer than after the −86S treatment; however those few floating cancer cells also exhibited several hundred-fold increase of the S-ncrRNA. Although the mechanism of upregulation is unclear, the S-ncrRNA may possibly act as a cell death regulator. Flow cytometric assays to detect fragmented DNA in cells after 24-hr and 48-hr treatments showed an apparent increase in apoptosis (2- to 4-fold) for the −86S treatment compared to −84AS and scrambled control. Further morphological analysis by transmission electron microscopy identified large and abundant autophagosomes and vacuoles preferentially in cancer cells, suggesting another possible cell death mechanism mediated by the oligonucleotide targeting AS-ncrRNA. In sum, rRNA processing and cis bidirectional noncoding rRNAs may form an interconnected network to maintain tumor phenotype. Interruption of this network by oligonucleotides targeting AS-ncrRNA activates massive elevation of the S-ncRNA level and sensitizes cancer cells preferentially for apoptotic and autophagic cell death. Targeting of AS-ncrRNA could have potential for human lung cancer therapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4085.