Abstract 1097: Knockdown of splicing factor ESRP1 affects multiple splicing factors

Abstract

Abstract Introduction: Tissue-specific alternative splicing (AS) is an important mechanism for regulating gene expression. Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2), two RNA-binding proteins (RBPs) that promote splicing, are potential candidates that contribute to breast cancer recurrence and resistance to therapies. In our prior studies, we have shown that ESRPs are associated with endocrine resistance. In this study, we seek to investigate the impact of the ESRP1 knockdown in endocrine resistant breast cancer cells. Methods: Expression of ESRP1 was analyzed in endocrine resistant-LCC2 and LCC9 cells. Knockdown of ESRP1 expression in LCC2 and LCC9 cells was achieved using lenti-viral based shRNA approach (Mission TRC human shRNA constructs, Sigma). We next performed paired-end RNA-sequencing of LCC2 and LCC9 cells transfected with control and two clones representative of shRNA knockdown (Illumina HiSeq platform, SeqWright Genomic Services) according to the manufacturer's instructions. After processing and quality check of raw FASTQ data (Illumina CASAVA, FASTQ and FASTX toolkits), reads were aligned, and quantified using bioinformatics software tools (Tophat/Cufflinks and Sailfish). Results: Expression of ESRP1 is significantly elevated in ERα-positive endocrine -resistant cells (LCC2 and LCC9 cells). ESRP1 knockdown was confirmed using qRT-PCR analysis in LCC2 cell lines (clone1 - 80% reduction and clone 3- 95% reduction) and in LCC9 cells (clone 2- 90% reduction and clone 3- 50% reduction). RNA-seq analysis identified 5153 differentially regulated genes between ESRP1 knockdown and control LCC2 cell lines. Of the 5153 genes, 1117 and 2109 genes were unique to clone 1 and clone 3, respectively, while 1927 genes were common in both clones. Targeted RNA-seq analysis also revealed that ESRP1 regulates the expression of several genes including other splicing factors. Furthermore, a functional enrichment analysis of these genes revealed a possible role of altering important pathways such as cell-cell adhesion and cell signaling. Additionally, it was observed that neither ESR1 nor any of its downstream genes were affected by the knockdown of ESRP1. This data was similar to that obtained in knockdown of LCC9 cells. Conclusions: ESRP1 regulates splicing of multiple genes in ER+ breast cancer. The process is independent of ER pathway. Targeting splicing could be a novel modality for treating endocrine therapy resistant breast cancer. Citation Format: Yesim Gökmen-Polar, Yaseswini Neelamraju, Xiaoping Gu, Gouthami Nallamothu, Sarath C. Janga, Sunil Badve. Knockdown of splicing factor ESRP1 affects multiple splicing factors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1097. doi:10.1158/1538-7445.AM2015-1097