Abstract 994: Long noncoding RNAs generated from the BRCA1 pseudogene regulate genomic instability and homologous recombination repair
Authors: Han Y. Jane , Zhang Jing , Mason Jennifer M. , Prat Aleix , Yoshimatsu Toshio , Perou Charles M. , Kwon John , Kannanganattu Prasanth , Olopade Olufunmilayo I. 
Published: July 2016

Background: Genomic instability is frequently associated with cancer and can be indicative of a poor prognosis for breast cancer. While the significant role of BRCA1 in regulating DNA damage repair and checkpoint activation is well defined, little is known about the biological properties of the BRCA1 pseudogene (BRCA1P1) in neoplastic processes. The BRCA1P1 pseudogene was created by a recent evolutionary event, in which a partial duplication of the BRCA1 gene was followed by an insertion of a processed pseudogene of RPLP1P4.

Methods: To functionally annotate BRCA1P1, we conducted DNA-FISH, RNA-FISH, a luciferase reporter gene assay, bisulfite sequencing, microarray analysis, radiation-induced foci by immunofluorescence, I-SceI-induced double strand break repair assay, Caspase 3/7 assay, flow cytometry, qRT-PCR, and Western blot analysis. Depletion of BRCA1P1-lncRNA was achieved using LNA-antisense oligonucleotides (ASO), while overexpression of BRCA1P1 was attained using the CRISPR-On activation system.

Results: BRCA1P1 pseudogene is transcribed as a ∼1.6 kb unspliced and nucleus-retained long non-coding RNA (lncRNA) using a bidirectional promoter between BRCA1P1 and the neighbor of BRCA1 gene 1 (NBR1). In contrast to the BRCA1 promoter, which is sensitive to hypoxia and subject to CpG island methylation, the pseudogene promoter did not respond to hypoxia nor did it undergo epigenetic modification. Expression of the BRCA1P1 pseudogene varies among 16 breast cell lines with higher expression in some lines, due in part to chromosome 17 polysomy. Interference of BRCA1P1-lncRNA expression with ASO increased genomic instability and spontaneous DNA damage as indicated by an increased spontaneous 53BP1 and RAD51 foci. Breast cancer cells with BRCA1P1 depletion exhibited a defect in DNA damage repair and increased apoptosis in response to irradiation and DNA damaging drugs, respectively. I-SceI-based assays confirmed a decrease in homologous recombination (HR) repair in cells with BRCA1P1 silencing. Interestingly, inhibition of BRCA1P1-lncRNA increased mRNA and protein expression of BRCA1, perhaps to compensate for the HR defect in the cells. The effects of BRCA1P1 overexpression on regulating DNA repair processes are currently under investigation in cells with BRCA1 mutations.

Conclusion: These findings suggest a novel role for the BRCA1P1 pseudogene in regulation of DNA damage repair in human breast cancer cells. It's potential role as a therapeutic target remains to be explored.

Citation Format: Y. Jane Han, Jing Zhang, Jennifer M. Mason, Aleix Prat, Toshio Yoshimatsu, Charles M. Perou, John Kwon, Prasanth Kannanganattu, Olufunmilayo I. Olopade. Long noncoding RNAs generated from the BRCA1 pseudogene regulate genomic instability and homologous recombination repair. [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 994.