Abstract P4-04-02: RNA-seq reveals functional lncRNAs associated with estrogen-receptor status in breast cancer
Authors: Banerjee N , Chothani S , Kamalakaran S , Varadan V , Miskimen K , Vadodkar A , Abu-Khalaf M , Sikov W , Harris LN , Dimitrova N 
Published: August 2014

Background: Long non-coding RNAs (lncRNAs) are pervasively transcribed in the genome yet their role in human disease is not well understood. LncRNAs can have regulatory effects on coding mRNAs through a number of mechanisms, including repressing their sense-strand protein-coding partners. There is also emerging evidence that estrogen signaling affects the expression of a wide variety of non-coding functional RNA transcripts in addition to protein-coding transcripts. We performed an RNA-seq study to characterize changes in lncRNA expression and evaluate their association to estrogen receptor (ER) status and estrogen signaling.

Methods: We sequenced transcriptomes of core biopsy RNA from 45 breast tumors obtained from neoadjuvant clinical trials BrUOG 211A/211B. RNA was derived from biopsy samples obtained before exposure to run-in monotherapy with either nab-paclitaxel, bevacizumab or trastuzumab. Paired-end sequencing was performed using amplified total RNA with 74bp read length, yielding genome-wide transcriptomic data. Transcriptomic abundance and differential expression were estimated assuming Poisson-distributed read-counts. Paired-end sequence data was aligned to a lncRNA database containing 14,572 unique lncRNAs. Changes in relative abundance of lncRNA transcripts were tested for association with estrogen receptor status using the Wilcoxon rank-sum test. Expression levels of the ER-associated lncRNAs were investigated in RNA-seq data from ER-positive MCF7 cells in response to treatment with E2 and tamoxifen (3hr, 12 hr and a non-treated control). ER-responsive binding sites on or near the ER-associated lncRNAs were investigated in a ChIP-seq study in MCF7 cells following estrogen treatment. MCF7 cell-line RNA-seq and ChIP-seq data were obtained from publicly available Short Read Archive (ERX030990 and SRX113365) at NCBI.

Results: On average, in each patient 5000 lncRNAs were detectable. Expression of 22 lncRNAs were associated ER status (p&lt;0.05) in breast tumors. 17 of the 22 ER-associated lncRNAs were detectable in ER-positive MCF7 breast cancer cells and were all responsive to E2 (estradiol) treatment. Interestingly, all 17 lncRNAs showed significant downregulation (&gt; = 2 fold) upon tamoxifen treatment. To further evaluate the regulatory relationship between ER and the 22 lncRNAs, we identified the binding sites of ER in estrogen-treated MCF7 cells using ChIP-seq. We found that &gt;50% of the ER-associated lncRNAs had ER binding site either overlapping or neighboring the lncRNA.

Discussion: We have shown that lncRNA expression levels are associated with ER status in breast tumors. We have further established that they are estrogen-responsive with a majority being direct targets of ER using cell-line data. Further functional validation studies are ongoing. We are also exploring lncRNA-mRNA expression for coding partners of lncRNAs to identify coding/noncoding gene regulatory networks important in estrogen response. Understanding the regulatory effects of lncRNA expression opens up new opportunities for stratification and management of breast cancers.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-04-02.