Abstract PD7-04: Fibroblast growth factor receptor 1 associates with promoters genome-wide and regulates gene transcription in ER+/FGFR1-amplified breast cancer: Implications for endocrine resistance

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 and alter gene expression through binding to chromatin. However, the mechanisms underpinning nuclear FGFR1-mediated gene transcription remain unclear. Thus, we sought to elucidate mechanisms to explain the genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer.Results: FGFR1 ChIP-Seq detected 4408 DNA binding sites in CAMA1 ER+/FGFR1-amplified breast cancer cells cultured in estrogen-free conditions; 67% of these sites were enriched at promoter regions, suggesting a role of FGFR1 in gene transcription regulation. ChIP-qPCR assay confirmed FGFR1 binding to promoter regions of genes such as CCND1, MYC, VEGFA, JUNB and SMAD5 in both CAMA1 and MDA-MB-134 ER+/FGFR1-amplified cells and also in an ER+/FGFR1-amplified patient derived xenograft (HCI-011). RNA-Seq of CAMA1 cells revealed that expression of FGFR1-bound genes was substantially higher than non FGFR1-bound genes (p<0.0001), suggesting FGFR1 binds to genes that are actively transcribed. Consistent with these results, precipitation with a FGFR1 antibody followed by immunoblot analysis showed association of FGFR1 with RNA Polymerase II (Pol II) in CAMA1, MDA-MB-134 and HCI-011 cell extracts. FGFR1 mainly bound with Pol II phosphorylated in Ser5 (Pol II S5P), a post-translational modification required for transcriptional activity. ChIP-Seq in CAMA1 cells with a Pol II S5P antibody revealed that 2867 of 4408 (65%) FGFR1 binding sites overlapped with Pol II S5P peaks, with a distribution centered on a similar location near the transcription start site. This interaction was validated by ChIP-reChIP assay, via sequential immunoprecipitation of FGFR1 and Pol II. Analysis of the METABRIC cohort showed that 1096/4408 (25%) FGFR1 DNA binding sites overlapped with genes differentially expressed in FGFR1-amplified vs FGFR1 non-amplified ER+ breast cancers. From this 1096-overexpressed gene list and using Gene Set Variation Analysis (GSVA), we developed a signature score for the top 102 genes (LogFC>0.25), representing those whose expression is likely regulated by FGFR1. This high signature score was associated with worse disease free survival (DFS; 263.7 months vs not reached; HR=1.72, CI 1.39-2.12; p<0.0001) and overall survival (OS; 145.1 vs 174.1 months; HR=1.24, CI 1.07-1.43; p=0.0003) in the ER+/HER2− cohort in METABRIC. This high signature score also correlated with high tumor grade (p<0.0001) and a worse Nottingham prognostic index (p<0.0001). Finally, we investigated cofactors influencing FGFR1 genomic function. Since nuclear FGFR1 has been shown to interact with ERα, we examined those cofactors involved in ER transcription. We initially focused on the FOXA1 pioneer factor, which mediates transcription factor binding to chromatin in ER+ breast cancer cells. Precipitation with a FGFR1 antibody followed by FOXA1 immunoblot analysis demonstrated an association of FGFR1 with FOXA1 in CAMA1 and MDA-MB-134 cells. ChIP in CAMA1 cells revealed FOXA1 enrichment at promoter regions bound by FGFR1. Further, siRNA-mediated FOXA1 knockdown in CAMA1 cells markedly reduced FGFR1 binding to several promoter regions, preliminarily including CCND1, JUNB, SMAD5, MYC and TOB1, as measured by ChIP-qPCR. Conclusions: These findings suggest a prominent role of FGFR1 in gene transcription regulation in breast cancer. Whether this transcriptional action is causal to antiestrogen resistance in ER+/FGFR1-amplified breast cancer is under active investigation and will be reported at the Symposium. Citation Format: Alberto Servetto, Rahul Kollipara, Luigi Formisano, Kyung-min Lee, Dhivya R Sudhan, Ariella B Hanker, Sumanta Chatterjee, Albert Lin, Saurabh Mendiratta, Nicholas James, Ralf Kittler, Carlos L Arteaga. Fibroblast growth factor receptor 1 associates with promoters genome-wide and regulates gene transcription in ER+/FGFR1-amplified breast cancer: Implications for endocrine resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD7-04.