Abstract 4402: FGFR1 signaling modulates estrogen-independent ER transcriptional activity in ER+/FGFR1-amplified breast cancer cells

Abstract

Abstract Background: FGFR1 amplification occurs in about 15% of estrogen receptor-positive (ER+) breast cancers and is associated with resistance to endocrine therapy. In these tumors, nuclear FGFR1 has been shown to interact with ERα. In addition, FGFR1 has been demonstrated to alter gene expression through binding to chromatin. However, the mechanisms underpinning nuclear FGFR1-mediated gene transcription remain unclear. Thus, we sought to elucidate the genomic and non-genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer. Methods: FGFR1 ChIP-Seq and ERα ChIP-Seq were performed on CAMA1 ER+/FGFR1-amplified breast cancer cells. ChIP-qPCR was employed to quantify DNA binding events. For ChIP-Seq, immunoblot, RT-qPCR, Estrogen Response Element (ERE) luciferase reporter and growth assays, CAMA1 cells were plated in estrogen-free media for 24 hours and then stimulated with 100 ng/ml FGF3 or 1 nM β-Estradiol. Results: FGFR1 ChIP-Seq detected 2211 DNA binding sites in CAMA1 cells cultured in estrogen-free conditions. Gene Set Enrichment Analysis (GSEA) revealed that the TNFα signaling via NF-KB, MYC targets, G2M checkpoints, ERE early and ERE late response genes (all FDR <0.00001) were among the most enriched gene sets. The majority of binding sites occurred in promoter regions, supporting a role of FGFR1 in regulation of gene transcription. FGFR1 ChIP-qPCR confirmed FGFR1 binding to promoter regions of oncogenes including CCND1, MYC, VEGFA, JUNB and SMAD5. FGF3 stimulation of CAMA1 cells further enriched FGFR1 binding to the CCND1 promoter and upregulation of CCND1 mRNA and protein levels. These effects were ablated upon addition of the pan-FGFR tyrosine kinase inhibitor rogaratinib. Motif analysis revealed CTCF (CCCTC binding factor) as the most enriched motif (p=1e-91). siRNA-mediated knockdown of CTCF inhibited FGF3-induced ERα transcriptional activity and proliferation of CAMA1 cells. These results suggest a role for CTCF in mediating the transcriptional programs regulated by FGFR1. We reported an association of nuclear FGFR1 and ERα in ER+/FGFR1-amplified breast cancer cells. Thus, we next investigated the role of FGF3-induced FGFR signaling on estrogen-independent ERα transcription using ERα ChIP-Seq. FGF3 stimulation of CAMA1 cells resulted in 407 DNA binding sites of which 155 were unique compared to cells in the absence of ligand. GSEA of these 155 peaks revealed enrichment for ERE early (p=3.08e-17) and ERE late (p=2.6e-5) response genes. FGF3-mediated induction of ERα transcriptional program was confirmed by ERE reporter assay and was abrogated by treatment with rogaratinib. Conclusions: These findings suggest a FGFR1 kinase-dependent role on ER-mediated transcription in ER+/FGFR1-amplified breast cancer cells. We are currently performing mass spectrometry analysis to identify binding partners of nuclear FGFR1 that mediate its transcriptional function. Citation Format: Alberto Servetto, Luigi Formisano, Rahul Kollipara, Dhivya R. Sudhan, Kyung-min Lee, Sumanta Chatterjee, Ariella B. Hanker, Saurabh Mendiratta, Ralf Kittler, Carlos L. Arteaga. FGFR1 signaling modulates estrogen-independent ER transcriptional activity in ER+/FGFR1-amplified breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4402.