Abstract 158: The p16/ARF intron 2 imbedded long non-coding RNA CAI2 is highly expressed in tumor cells and modulates chromatin structure

Abstract

Abstract Over the last several years it has become apparent that the majority of the genome is transcribed with the number of non-coding RNA (ncRNA) genes significantly higher than the number of protein-encoding genes. The 9p21 locus harbors a number of tumor suppressor genes including p15, p10, ARF, chARF, p16, p16γ, p12 and p16-ACT, and the long ncRNAs ANRIL and p15AS. The abundance of regulatory genes in such a short span perhaps accounts for why this region is frequently inactivated in many cancers. We report on the novel lncRNA CAI2, a non-conserved gene imbedded in p16/ARF intron 2 with homology that is limited to the upper primates. CAI2 is a single exon, RNA pol II regulated gene with a poly A signal localized in p16/ARF exon 3, yet independent of the p16/ARF poly A signal. The expression levels of CAI2, p16 and ARF genes are highly correlated, being low in normal tissue but high in most tumor cell lines with an intact 9p21 locus. Notably, high levels of CAI2 expression are associated with advanced-stage of neuroblastoma, one of the few tumor types where 9p21 alterations are infrequent and where p16 is paradoxically overexpressed. CAI2 expression was associated with a poor outcome in neuroblastoma, suggesting value as a biomarker of tumor cell stage and patient outcome. Despite the strong correlation between CAI2 and p16 expression, CAI2 is independently regulated and is expressed in cell lines deleted or epigenetically silenced for p16, ARF and even p15. In response to serum deprivation, CAI2 levels rise rapidly in normal mononuclear cells, but slowly in tumor cell lines, suggesting that CAI2 is also a stress-response gene. CAI2 levels also rise in response to chemotherapeutic insult such as etoposide treatment. The NMB7 neuroblastoma cell line is one of very few neuroblastoma cell lines to express CAI2 at low levels. During passage, this cell line undergoes morphological changes, proliferates faster and exhibits an increase of more than 100-fold in CAI2 expression, though a concomitant decrease in Tyrosine Hydroxylase expression suggests the changes are not due to differentiation. Consistent with this, Retinoic Acid treatment, part of standard neuroblastoma therapeutic regimen known to induce neuroblastoma cell differentiation, slightly decreased CAI2 expression. As many lncRNAs regulate chromatin states, we considered epigenetic roles for CAI2 in gene regulation. RNA Immunoprecipitation revealed that CAI2 interacts with H3K27me3, a recruiter of Polycomb Repressive Complex 1 (PRC1) that modulates chromatin structure and gene expression. As many advanced stage neuroblastomas and neuroblastoma cell lines highly express p16, we speculate that CAI2 binding to H3K27me3 may modulate chromatin structure and regulate expression at the 9p21 locus. Citation Format: Olga Cohen, Alice Yu, Mitchell B. Diccianni. The p16/ARF intron 2 imbedded long non-coding RNA CAI2 is highly expressed in tumor cells and modulates chromatin structure. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 158. doi:10.1158/1538-7445.AM2015-158