Abstract 980: The long non-coding RNA CAI2 is a tumor suppressor gene
Authors: Cohen Olga , Lin Ruey-Jen , Yu Alice L. , Diccianni Mitchell B. 
Published: July 2016

Abstract
We have recently reported on the discovery of a long non-coding RNA imbedded in intron 2 of the p16/ARF (CDKN2A) locus at 9p21. CAI2 is a non-conserved, RNA pol II regulated gene with whose expression is highly correlated with parent genes p16 and ARF. However, CAI2 is expressed in cell lines deleted for p16, ARF and even p15, and is expressed cell lines epigenetically silenced for p16 and/or ARF. Furthermore, treatment of p16 and/or ARF epigenetically silenced cell lines with 5-aza-2’-deoxycytidin allows p16 and/or ARF re-expression while having no influence on CAI2, confirming independent regulation. Speculating, therefore, that the regulatory region of CAI2 lies imbedded in the non-transcribed portion of intron 2, we created expression constructs of the immediate 5’ region of CAI2. However, consistent with a bioinformatical analysis, no regulatory activity was detected. This suggests that this non-coding RNA is non-traditionally controlled by remote but yet to be identified regulatory element(s) and/or mechanisms. By colony formation assay, CAI2 overexpression can inhibit cell growth in the p16 intact HEK293T and SK-BR-3 breast cancer cell lines. RNA Immunoprecipitation has revealed that CAI2 interacts with H3K27me3, a recruiter of Polycomb Repressive Complex 1 (PRC1) that modulates chromatin structure and gene expression, and BMI1, a PRC1 complex protein that is a negative regulator of p16. Consistent with this effect, CAI2 can induce p16 expression in HEK293T cells, and this was hypothesized to be the mechanism by which CAI2 inhibits cell growth. However, we now show that CAI2 can also inhibit colony formation in the p16/ARF deleted MCF7 and MDA-MB-231 breast cancer cell lines. Interestingly however, CAI2 overexpression had little to no effect on 2D cultures of HEK293T or MCF7 cells including DNA synthesis, XTT metabolic activity or cell count.
As CAI2 is imbedded in the p16/ARF gene, standard siRNAs may inadvertently influence p16 and/or ARF expression due to actions on their pre-mRNAs. Therefore, we created a series of partial CAI2 knockouts using CRISPR technology and compared their growth by colony formation assay. Notably, clones containing a partial knockout of the 5’ region of CAI2 proliferated faster than mock transfected clones, consistent with the growth suppressive properties observed with CAI2 overexpression. In 2D cultures, a small increase in DNA synthesis was also observed in the partial deletions. The HEK293T clones containing partial deletions of CAI2 also exhibited small increases in expression of the remaining intact portions of the gene with a concomitant decrease in p16 expression, all consistent with an influence of CAI2 on p16 expression and cell growth; no influence on ARF expression was observed.
Taken together, the data demonstrate that CAI2 is capable of influencing p16 expression, but this influence is insufficient to fully account for the ability of CAI2 to regulate growth, and we conclude that CAI2 is a bona fide tumor suppressor gene in its own right.
Citation Format: Olga Cohen, Ruey-Jen Lin, Alice L. Yu, Mitchell B. Diccianni. The long non-coding RNA CAI2 is a tumor suppressor gene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 980.