Abstract S4-05: Interrogating the landscape of long noncoding RNAs in breast cancer to identify predictors of tamoxifen resistance
Authors: Feng FY , Niknafs Y , Han S , Ma T , Speers C , Malik R , Evans J , Zhang C , Pierce LJ , Hayes DF , Rae JM , Chinnaiyan AM 
Published: May 2016

Background: We previously performed an informatics-based analysis on RNA sequencing libraries from 7,256 tumor and normal tissue specimens 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 MK 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 prioritize differentially expressed lncRNAs in cancer vs normal tissue, and in ER+ vs ER- disease, we performed Sample Set Enrichment Analysis (SSEA) on &gt;1000 RNA Seq libraries, from breast cancer and normal tissue samples from The Cancer Genome Atlas project. The effect of the top prioritized lncRNA on cancer phenotypes was studied via in vitro proliferation, colony formation, invasion and tamoxifen resistance assays in MCF7 and T47D cells, and via in vivo mouse xenograft studies and chick chorioallantoic membrane (CAM) assays. To study the mechanism by which this lncRNA promotes tumor progression, we identified its top protein interactors and subdomains responsible for function, and then studied the effects of disrupting function of this lncRNA on cancer phenotypes. Finally, in a "guilt-by-association" study, we developed a signature of 150 protein coding genes most strongly associated with our lncRNA of interest, and investigated the association of this signature with clinical outcomes using Oncomine analyses.

Results: SSEA analysis on over 1000 TCGA samples nominated Breast Cancer Associated Transcript (BRCAT 431) as the top overexpressed ER-regulated lncRNA in breast cancer. In vitro experiments demonstrate that siRNA-mediated knockdown of BRCAT431 resulted in significantly decreased proliferation, colony formation, and invasion (by &gt;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. In vivo xenograft and CAM studies demonstrate that knockdown of BRCAT431 also significantly decreased xenograft growth and tumor invasion by &gt;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 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 compendia 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. Our results suggest that by promoting tamoxifen resistance, BRCAT431 increases the clinical risk of recurrence and metastases in breast cancer. Overall, this study supports the rationale for investigating lncRNAs as novel biomarkers and therapeutic targets in breast cancer.

Citation Format: Feng FY, Niknafs Y, Han S, Ma T, Speers C, Malik R, Evans J, Zhang C, Pierce LJ, Hayes DF, Rae JM, Chinnaiyan AM. Interrogating the landscape of long noncoding RNAs in breast cancer to identify predictors of tamoxifen resistance. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-05.