De novo or acquired resistance to endocrine therapy limits its utility in a significant number of estrogen receptor (ER) breast cancers. An increasing number of molecular assays predict the likelihood of distant recurrence in tamoxifen-treated patients with node-negative, ER+ breast cancer. However, these do not provide the mechanistic basis for endocrine resistance. It is crucial to identify novel targets and improve the success of endocrine therapies. We previously shown that epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2), RNA binding proteins that promote splicing, are significantly elevated in cases with high Oncotype DX scores (innate resistance) and in ERα-positive cells with acquired tamoxifen resistance (MCF-7 LCC2 cells) and fulvestrant and tamoxifen resistance (MCF-7/LCC9 cells). The aim of this study was to investigate the ESRP1/ESRP2-regulated alternative splicing events leading to innate and acquired endocrine resistance.
A combinatorial bioinformatics approach was employed to identify genes altered through alternative splicing by ESRP1/ESRP2 in endocrine resistance. Briefly, genes with ESRP1/ESRP2 motifs were screened in the human genome. This data was integrated with tamoxifen-treated datasets, and filtered using protein-protein interactions (PPI; BIOGRID) and clinical outcome (KM plotter. Potential target gene transcripts were further narrowed down using an algorithm based on motif binding location and the Cancer Genome Atlas (TCGA) breast cancer datasets with low and high ESRP1 cases. The alternative transcripts were further validated using splice variant specific-custom qRT-PCR in low and high RS Oncotype cases as surrogate for endocrine sensitivity and resistance.
Motif analysis in combination with tamoxifen-treated datasets identified 2212 differentially expressed genes. Further collective analysis of number of PPI (>50) and survival data from KM plotter (P<0.001) narrowed down 79 candidate genes that are associated with tamoxifen resistance. TCGA data confirmed presence of distinct transcripts (splicing variants) based on ESPR1 expression levels. Splice specific RT-PCR confirmed alternative splicing events involving cycle related genes such as AURKA, FZR1, and MDM2 in low ESRP1 (and low RS) and high ESRP1 (high RS) cases.
ESRP1/ESRP2 by inducing alternative splicing play an important role in tamoxifen resistance and recurrence of ER+ breast cancer. Targeting alternative splicing may offer novel avenues for combating endocrine-resistance in breast cancer.
Citation Format: Yesim Gokmen-Polar, Yaseswini Neelamraju, Chirayu Pankaj Goswami, Harikrishna Nakshatri, Sarath Chandra Janga, Sunil Badve. ESRP1 adds sp(l)ice to endocrine resistance [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 P3-05-20.