Abstract P5-04-03: Aggressiveness of epithelial cancers is independent of epithelial-to-mesenchymal transition
Authors: Badve SS , Neelamraju Y , Goswami CP , Gu X , Nallamothu G , Gu Y , Vieth E , Janga SC , Ryan M , Gokmen-Polar Y 
Published: February 2018

Background: Epithelial-to-Mesenchymal Transition (EMT)is postulated to be an important step in cancer progression and controlled by multiple mechanisms including EMT transcription factors (EMT-TFs) and splicing factors such as Epithelial Splicing Regulatory Proteins (ESRP1 and ESRP2). We previously have shown that the expression of ESRP1 and ESRP2 have significantly elevated in cases with high Oncotype DX scores and in ERα-positive cells with acquired endocrine resistance (SABCS 2013). This study seeks to identify the role of EMT-TFs and ESRP1s in the determination of outcomes of patients with ER+ breast cancer.

Patients and Methods: The expression of EMT-TFs and ESRP1 was analyzed in the Affymetrix microarray and TCGA BRCA databases. Next, we generated genetically engineered knockdown models of ESRP1 to understand its functional role in endocrine resistance. We performed RNA-seq and MATS analysis to identify alternative splicing events (ASEs) between ESRP1 knockdown and control cell lines [(2C3 vs 2-control (LCC2 set) and 9C2 vs 9-control(LCC9 set)]. Validation of the ASEs was performed using a probe-based platform [Human Transcriptome Array 2.0 (HTA)] and TCGA SpliceSeq from breast tumors.

Results: High levels of ESRP1 mRNA, but not EMT-TFs, are associated with poor prognosis in human ER+ breast tumors (Affymetrix; P=2.8e-07 and TCGA; P=0.00011). Knockdown of ESRP1 in ER+ endocrine resistant breast cancer induced glandular differentiation, rather than mesenchymal features. This was associated with significant reduction in cell and tumor growth in mammary fat pad orthotopic xenograft mice models of LCC2 and LCC9. No alterations in EMT-TFs were observed in these cells. Transcriptome profiling of ESRP1 knockdown cells further revealed altered ASEs in EMT splicing gene signature, but not at the gene level. These alterations (SE-skipped exon) were further validated for ARHGEF11, ENAH, FNIP1, SCRIB, and SLK using probe based HTA platform for ESRP1 knockdown cells and TCGA-SpliceSeq ER+ BRCA tumors in ER+ ESRP1low versus ESRP1high breast tumors.

Conclusions: Our data demonstrates for the first time that high ESRP1 is associated with poor prognosis in ER+ breast cancer. Despite its involvement in regulation of EMT splicing signature, low levels (or knockdown) of ESRP1 were not associated with EMT phenotype in tumors or in endocrine-resistant ER+ cells. Taken together, our findings indicate that EMT is not important in determining prognosis in ER+ breast cancer and that ESRP1 exerts a different role in aggressive ER+ breast cancers.

Citation Format: Badve SS, Neelamraju Y, Goswami CP, Gu X, Nallamothu G, Gu Y, Vieth E, Janga SC, Ryan M, Gokmen-Polar Y. Aggressiveness of epithelial cancers is independent of epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-04-03.