Abstract 2662: Interrogation of the landscape of long noncoding RNAs in breast cancer to identify an ER-regulated predictor of tamoxifen resistance

Abstract

Abstract Background: We previously performed a bioinformatics-based analysis on 7,256 RNA sequencing libraries from tumors, normal tissues, and cell lines to delineate the landscape of long noncoding RNAs (lncRNAs) in the human transcriptome. This analysis identified 58,648 lncRNAs, including over 45,000 novel transcripts (Iyer and Niknafs et al, Nature Genetics, 2015). We now interrogate this lncRNA compendium to identify top candidate estrogen receptor (ER)-associated lncRNAs in breast cancer and characterize their association with disease progression. Methods: To identify and prioritize lncRNAs that were differentially expressed in cancer vs normal tissue, and in ER-positive vs ER-negative disease, we performed differential expression testing on over 1000 RNA sequencing libraries including breast cancer and normal tissue samples from the The Cancer Genome Atlas (TCGA) project. The effect of the top prioritized lncRNA on cancer phenotypes was studied via proliferation, colony formation, invasion and tamoxifen resistance assays in MCF7 and T47D cell lines, and via mouse xenograft studies. The mechanism by which this lncRNA promotes tumor progression was investigated by identifying its top protein interactors and its subdomains responsible for function, and then studying the effects of disrupting function of this lncRNA on cancer phenotypes. Results: Differential expression analyses nominated BRCAT431 as the top overexpressed ER-regulated lncRNA in breast cancer. In vitro experiments demonstrate that shRNA-mediated knockdown of BRCAT431 significantly decreased proliferation, colony formation, and invasion (by >50% in most assays). Tamoxifen resistance was associated with significantly increased BRCAT431 levels in both MCF7 and T47D cells, and knockdown of BRCAT431 reversed tamoxifen resistance. Xenograft studies demonstrate that knockdown of BRCAT431 also significantly decreased xenograft growth and tumor invasion by >50%. RNA pulldown followed by mass spectrometry identified the RNA binding protein hnRNPL as a key protein interacting with BRCAT431. Deletion studies identified a 27 base-pair region of BRCAT431 necessary for its interaction with hnRNPL, and loss of this region abrogated BRCAT431-induced invasion. Finally, guilt-by-association studies demonstrate a strong association between BRCAT431 overexpression and tumor grade, recurrence, and metastases. Conclusion: In this study, we develop the largest reported compendium of breast cancer lncRNAs. We prioritize BRCAT431 as the top lncRNA upregulated in ER-positive breast cancers, and demonstrate that it confers aggressive oncogenic phenotypes in vitro and in vivo. We identify a novel mechanism by which this lncRNA functions through interaction with hnRNPL. Our results suggest that by promoting tamoxifen resistance, this lncRNA increases the clinical risk of recurrence and metastases in breast cancer. Citation Format: Yashar Niknafs, Sumin Han, Teng Ma, James Rae, Arul Chinnaiyan, Felix Feng. Interrogation of the landscape of long noncoding RNAs in breast cancer to identify an ER-regulated predictor of tamoxifen resistance. [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 2662.