Abstract 1304: FGFR1 associates with gene promoters and regulates transcription in ER+/FGFR1-amplified breast cancer: Implications for endocrine resistance

Abstract

Abstract Background: FGFR1 amplification occurs in ~ 15% of estrogen receptor-positive (ER+) breast cancers. In these tumors, nuclear FGFR1 has been shown to interact with DNA, but its engagement in transcription regulation remains unclear. Thus, we investigated the mechanisms underpinning the genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer. Methods: FGFR1-ChIP-Seq was performed in CAMA1 ER+/FGFR1-amplified human breast cancer cells to identify genomic distribution of FGFR1. IP with FLAG antibody followed by Mass Spectrometry (MS) was carried out on nuclear plus chromatin fractions of CAMA1 cells overexpressing 3XFLAG-FGFR1 to uncover the nuclear FGFR1 interactome. Results: FGFR1-ChIP-Seq detected 4408 peaks in CAMA1 cells cultured in estrogen-free conditions, with marked enrichment of GC-rich consensus motifs. 67% of peaks were enriched at promoter regions. ChIP-PCR confirmed FGFR1 binding to several genomic loci in ER+/FGFR1-amplified cell lines (CAMA1 and MDA-MB-134) and PDX (HCI-011). Further, MS uncovered RNA Polymerase II subunits among the top nuclear FGFR1 interacting proteins. FGFR1 mainly bound Pol II phosphorylated on Ser5 (Pol II S5P), a marker of transcription initiation, in CAMA1, MDA-MB-134 and HCI-011 cell extracts. Pol II S5P-ChIP-Seq revealed that 65% (2867/4408) of FGFR1 peaks were shared with Pol II S5P in CAMA1 cells. Also, ChIP-Seq revealed that 95% of FGFR1 peaks overlapped with both H3K4me3 and H3K27ac, markers of active transcription. Consistent with these results, RNA-Seq of CAMA1 cells showed that expression of FGFR1-bound genes was markedly higher than non FGFR1-bound genes (p<0.0001), suggesting that FGFR1 binds to actively transcribed genes. The pioneer factor FOXA1 was detected by MS among FGFR1 interacting proteins and ChIP-Seq showed its enrichment at FGFR1-bound promoters. Notably, siRNA-mediated FOXA1 knockdown reduced FGFR1 distribution to several genomic loci in CAMA1 cells, as measured by FGFR1-ChIP-Seq. Finally, we found that 10% (360/3558) of FGFR1 peaks overlapped with genes that were differentially expressed in FGFR1-amplified (n=7) vs FGFR1 non-amplified (n=30) ER+ breast tumors treated with neoadjuvant letrozole. Using Gene Set Variation Analysis, we developed a signature score for the top 32/360 genes (LogFC>1), whose expression is likely regulated by nuclear FGFR1. A high signature score correlated with resistance to letrozole (p<0.0001) and high post-treatment Ki67 index (p=0.0004) in our cohort, and with worse disease-free and overall survival in the METABRIC cohort of ER+/HER2− patients treated with endocrine therapy. Conclusions: These findings support a prominent role for FGFR1 in the transcriptional machinery of breast cancer cells. Whether this transcriptional action is causal to antiestrogen resistance in ER+/FGFR1-amplified breast cancer is currently under investigation. Citation Format: Alberto Servetto, Rahul Kollipara, Luigi Formisano, Kyung-min Lee, Albert Lin, Dhivya R. Sudhan, Ariella B. Hanker, Sumanta Chatterjee, Hiroaki Akamatsu, Arnaldo Marin, Saurabh Mendiratta, Ralf Kittler, Carlos L. Arteaga. FGFR1 associates with gene promoters and regulates transcription in ER+/FGFR1-amplified breast cancer: Implications for endocrine resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1304.