Abstract P3-05-18: RNA-seq reveals lncRNAs associated with hormonal influences and endocrine therapy
Authors: Banerjee Nilanjana , Verhaegh Wim , Cheung Patrick , Dimitrova Nevenka 
Published: May 2015

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 hormone signaling affects the expression of a wide variety of lncRNAs. However, the role of lncRNAs in the response mechanisms to endocrine therapy remains mostly unknown. We investigated MCF7 and T47D cell-line RNA-seq data to characterize changes in lncRNA expression and evaluate their response to estradiol (E2), progesterone (P4) and to their antagonists, tamoxifen (TAM) and mifepristone (RU486).

Methods: RNA-seq data from MCF7 cell-lines with response to E2 and TAM and T47D cell-lines with response to P4 and RU486 were obtained from publicly available short read archive (SRA: ERP000992). Expression values after hormonal or antagonist treatments were compared to non-treated cells. We independently sequenced total RNA samples of T47D cell-lines with or without E2 treatment and created a robust list E2-responsive lncRNAs for further analysis. The RNA-sequencing data was aligned to a lncRNA database containing 14,572 unique lncRNAs. Significant log-fold changes in relative abundance of lncRNA transcripts were selected using percentile-p-value. ER-responsive binding sites on or near the ER-associated lncRNAs were investigated in a ChIP-seq study in MCF7 cells following estrogen treatment. Finally, we identified the subset of hormonally-responsive lncRNAs that are also evolutionarily conserved.

Results: On average, in each cell-line experiment, 5000 lncRNAs were detectable. Expression of 55 lncRNAs were associated with differing response to E2 and TAM (p&lt;0.01) while 120 lncRNAs were associated with differing response to P4 and RU486 (p&lt;0.01). To probe whether similar lncRNA-related response mechanisms are at play for both types of hormonal influences, we examined the overlap between the two sets of significantly responsive lncRNAs (&gt;3-fold change between response to agonist and antagonist). Less than 10% of the lncRNAs were common among the two sets suggesting that while there might be some cross-talk, E2 and P4 are generally regulating unique sets of lncRNAs. For example, XLOC_013954, a bona-fide E2-responsive lncRNA shows 5-fold difference between response to E2 and TAM, but has a fold change of mere 1.1 between response to P2 and RU486. From the MCF7 ChIP-seq data we found that &gt;50% of the ER-associated lncRNAs had ER binding site either overlapping or neighboring the lncRNA. Less than 30% of the response-associated lncRNAs were evolutionarily conserved suggesting they might be under different selection pressures.

Discussion: We have shown that lncRNA expression levels are associated with hormonal influences and endocrine therapy. We have further established that the lncRNAs impacted by the different hormonal influences are quite exclusive and suggest separate regulatory networks. We are also exploring lncRNA-mRNA expression for coding partners of the response-associated lncRNAs. Understanding the regulatory effects of lncRNA expression opens up new opportunities for stratification and management of breast cancers.

Citation Format: Nilanjana Banerjee, Wim Verhaegh, Patrick Cheung, Nevenka Dimitrova. RNA-seq reveals lncRNAs associated with hormonal influences and endocrine therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-18.